Modulation of PPAR-alpha expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of PPAR-alpha. The compositions comprise oligonucleotides, targeted to nucleic acid encoding PPAR-alpha. Methods of using these compounds for modulation of PPAR-alpha expression and for diagnosis and treatment of disease associated with expression of PPAR-alpha are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of PPAR-alpha. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding PPAR-alpha. Such compounds are shown herein to modulate the expression of PPAR-alpha.

BACKGROUND OF THE INVENTION

[0002] Steroid, thyroid and retinoid hormones produce a diverse array of physiologic effects through the regulation of gene expression. Upon entering the cell, these hormones bind to a unique group of intracellular nuclear receptors which have been characterized as ligand-dependent transcription factors. This complex then moves into the nucleus where the receptor and its cognate ligand interact with the transcription preinitiation complex affecting its stability and ultimately the rate of transcription of the target genes.

[0003] Peroxisome proliferators are a diverse group of chemicals which include hypolipidemic drugs, herbicides, leukotriene antagonists, and plasticizers, and are so called because they induce an increase in the size and number of peroxisomes. Peroxisomes are subcellular organelles found in plants and animals, and contain enzymes for respiration, cholesterol and lipid metabolism. The fibrate class of hypolipidemic drugs is used to reduce triglycerides and cholesterol in patients with hyperlipidemia, a major risk factor for coronary heart disease.

[0004] The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor subfamily of transcription factors. PPARs form heterodimers with other members of the nuclear hormone receptor superfamily and these heterodimers regulate the transcription of various genes. There are 3 known subtypes of PPARs, PPAR-alpha, PPAR-delta, and PPAR-gamma.

[0005] PPAR-alpha (peroxisome proliferator-activated receptor-alpha, also known as PPARA) was cloned from a human liver cDNA library and mapped to chromosome 22q12-ql3.1 in 1993 (Sher et al., Biochemistry, 1993, 32, 5598-5604). PPAR-alpha is mostly present in tissues characterized by high rates of fatty acid catabolism such as liver, kidney, heart and skeletal muscle where it regulates lipid metabolism and inhibits inflammatory response in the vascular wall (Chinetti et al., Inflammation Res., 2000, 49, 497-505; Fruchart et al., Curr. Opin. Lipidol., 1999, 10, 245-257). PPAR-alpha is also present in endothelial and smooth muscle cells, monocytes and monocyte-derived macrophages and its activation has been found to induce apoptosis in monocyte-derived macrophages (Fruchart et al., Curr. Opin. Lipidol., 1999, 10, 245-257).

[0006] Nucleic acid sequences encoding PPAR-alpha are disclosed and claimed in U.S. Pat. No. 5,685,596 and PCT publication WO 01/20037 (Hudson et al., 2001; Mukherjee, 1997).

[0007] Gervois et al. have described PPAR-alpha-tr, a truncated splice variant of PPAR-alpha which may negatively interfere with normal PPAR-alpha function (Gervois et al., Mol. Endocrinol., 1999, 13, 1535-1549). Five additional variants of the main mRNA of PPAR-alpha have been identified and are herein designated PPAR-alpha-2, PPAR-alpha-3, PPAR-alpha-4, PPAR-alpha-5 and PPAR-alpha-6.

[0008] In developed societies, metabolic disorders such as hyperlipidemia, athersclerosis, diabetes and obesity are usually part of a complex phenotype of metabolic abnormalities called syndrome X. Fibrates such as gemfibrozil, bezafibrate and fenofibrate are potent hypolipidemic drugs which bind to PPAR-alpha with high affinity, suggesting that the effects of fibrates on disease progression are mediated by PPAR-alpha (Kersten et al., Nature, 2000, 405, 421-424).

[0009] Methods using fatty acid CoA thioesters as small molecule inhibitors of PPAR-alpha have been disclosed and claimed in PCT publication WO 01/21181 (Murakami et al., 2001). Additionally, Kehrer et al. have found that MK886, an apoptosis-inducing inhibitor of 5′-lipoxygenase activating protein, also acts as an inhibitor of PPAR-alpha (Kehrer et al., Biochem. J., 2001, 356, 899-906).

[0010] Sartippour et al. have described the use of anti-PPAR-alpha antibodies in investigations of regulation of PPAR-alpha expression by glucose (Sartippour and Renier, Arterioscler. Thromb. Vasc. Biol., 2000, 20, 104-110).

[0011] Mice lacking the PPAR-alpha gene have been found to display a prologed response to inflammatory stimuli, indicating that PPAR-alpha has anti-inflammatory action (Kersten et al., Nature, 2000, 405, 421-424). More recent investigations of PPAR-alpha knockout mice have indicated enhanced hepatocyte proliferation in response to hepatomitogens, progressive dyslipidemia, sexually dimorphic obesity, steatosis, and disorders of fatty acid metabolism (Columbano et al., Hepatology (Philadelphia, Pa., U.S.), 2001, 34, 262-266; Costet et al., J. Biol. Chem., 1998, 273, 29577-29585; Djouadi et al., J. Clin. Invest., 1998, 102, 1083-1091; Leone et al., Proc. Natl. Acad. Sci. U.S. A., 1999, 96, 7473-7478).

[0012] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of PPAR-alpha. To date, investigative strategies aimed at modulating PPAR-alpha expression have involved the use of antibodies, small molecule agonists and antagonists and gene knock-outs in mice. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting PPAR-alpha function.

[0013] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of PPAR-alpha.

[0014] The present invention provides compositions and methods for modulating expression of PPAR-alpha, including modulation of variants of PPAR-alpha.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding PPAR-alpha, and which modulate the expression of PPAR-alpha. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of PPAR-alpha and methods of modulating the expression of PPAR-alpha in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of PPAR-alpha are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0016] A. Overview of the Invention

[0017] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding PPAR-alpha. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding PPAR-alpha. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding PPAR-alpha” have been used for convenience to encompass DNA encoding PPAR-alpha, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0018] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of PPAR-alpha. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0019] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0020] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0021] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0022] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0023] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0024] B. Compounds of the Invention

[0025] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0026] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0027] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0028] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0029] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0030] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0031] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0032] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0033] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0034] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0035] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0036] C. Targets of the Invention

[0037] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes PPAR-alpha.

[0038] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0039] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding PPAR-alpha, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0040] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0041] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0042] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0043] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0044] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0045] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0046] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0047] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0048] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0049] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0050] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0051] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0052] D. Screening and Target Validation

[0053] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of PPAR-alpha. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding PPAR-alpha and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding PPAR-alpha with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding PPAR-alpha. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding PPAR-alpha, the modulator may then be employed in further investigative studies of the function of PPAR-alpha, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0054] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0055] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0056] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between PPAR-alpha and a disease state, phenotype, or condition. These methods include detecting or modulating PPAR-alpha comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of PPAR-alpha and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0057] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0058] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0059] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0060] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0061] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0062] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding PPAR-alpha. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective PPAR-alpha inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding PPAR-alpha and in the amplification of said nucleic acid molecules for detection or for use in further studies of PPAR-alpha. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding PPAR-alpha can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of PPAR-alpha in a sample may also be prepared.

[0063] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0064] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of PPAR-alpha is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a PPAR-alpha inhibitor. The PPAR-alpha inhibitors of the present invention effectively inhibit the activity of the PPAR-alpha protein or inhibit the expression of the PPAR-alpha protein. In one embodiment, the activity or expression of PPAR-alpha in an animal is inhibited by about 10%. Preferably, the activity or expression of PPAR-alpha in an animal is inhibited by about 30%. More preferably, the activity or expression of PPAR-alpha in an animal is inhibited by 50% or more.

[0065] For example, the reduction of the expression of PPAR-alpha may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding PPAR-alpha protein and/or the PPAR-alpha protein itself.

[0066] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0067] F. Modifications

[0068] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0069] Modified Internucleoside Linkages (Backbones)

[0070] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0071] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0072] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0073] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside-linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0074] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0075] Modified Sugar and Internucleoside Linkages-Mimetics

[0076] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0077] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0078] Modified Sugars

[0079] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂) O] CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂) CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0080] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0081] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0082] Natural and Modified Nucleobases

[0083] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0084] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0085] Conjugates

[0086] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0087] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0088] Chimeric compounds

[0089] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0090] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0091] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0092] G. Formulations

[0093] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0094] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0095] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0096] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0097] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0098] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0099] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0100] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0101] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0102] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0103] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0104] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0105] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0106] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0107] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0108] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0109] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0110] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0111] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0112] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0113] H. Dosing

[0114] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0115] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0116] Synthesis of Nucleoside Phosphoramidites

[0117] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylamino-oxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0118] Oligonucleotide and Oligonucleoside Synthesis

[0119] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0120] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0121] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0122] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0123] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0124] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0125] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0126] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0127] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0128] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0129] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0130] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0131] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0132] RNA Synthesis

[0133] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0134] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0135] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0136] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0137] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0138] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0139] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5×annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0140] Synthesis of Chimeric Oligonucleotides

[0141] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0142] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0143] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′ dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0144] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0145] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites. [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0146] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0147] Design and Screening of Duplexed Antisense Compounds Targeting PPAR-alpha

[0148] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target PPAR-alpha. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0149] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0150] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0151] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate PPAR-alpha expression.

[0152] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 uL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 pL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0153] Oligonucleotide Isolation

[0154] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0155] Oligonucleotide Synthesis—96 Well Plate Format

[0156] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0157] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0158] Oligonucleotide Analysis—96-Well Plate Format

[0159] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0160] Cell Culture and Oligonucleotide Treatment

[0161] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0162] T-2-4 Cells:

[0163] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0164] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0165] A549 Cells:

[0166] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0167] NHDF Cells:

[0168] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0169] HEK Cells:

[0170] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0171] Primary Mouse Hepatocytes

[0172] Primary mouse hepatocytes were prepared from CD-1 mice purchased from Charles River Labs. Primary mouse hepatocytes were routinely cultured in Hepatoyte Attachment Media (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco/Life Technologies, Gaithersburg, Md.), 250 nM dexamethasone (Sigma), 10 M bovine insulin (Sigma). Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/well for use in RT-PCR analysis.

[0173] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0174] Treatment with Antisense Compounds:

[0175] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0176] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0177] Analysis of Oligonucleotide Inhibition of PPAR-Alpha Expression

[0178] Antisense modulation of PPAR-alpha expression can be assayed in a variety of ways known in the art. For example, PPAR-alpha mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0179] Protein levels of PPAR-alpha can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to PPAR-alpha can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0180] Design of Phenotypic Assays and In Vivo Studies for the Use of PPAR-Alpha Inhibitors

[0181] Phenotypic Assays

[0182] Once PPAR-alpha inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of PPAR-alpha in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0183] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with PPAR-alpha inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0184] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0185] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the PPAR-alpha inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0186] In Vivo Studies

[0187] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0188] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or PPAR-alpha inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a PPAR-alpha inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0189] Volunteers receive either the PPAR-alpha inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding PPAR-alpha or PPAR-alpha protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0190] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0191] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and PPAR-alpha inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the PPAR-alpha inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0192] RNA Isolation

[0193] Poly(A)+ mRNA Isolation

[0194] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0195] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0196] Total RNA Isolation

[0197] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0198] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0199] Real-Time Quantitative PCR Analysis of PPAR-Alpha mRNA Levels

[0200] Quantitation of PPAR-alpha mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0201] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0202] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0203] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0204] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0205] Probes and primers to human PPAR-alpha were designed to hybridize to a human PPAR-alpha sequence, using published sequence information (a genomic sequence of human PPAR-alpha represented by residues 58000-144000 of GenBank accession number NT_(—)011523.7, incorporated herein as SEQ ID NO: 4). For human PPAR-alpha the PCR primers were:

[0206] forward primer: GGCGATCTAGAGAGCCCGTTA (SEQ ID NO: 5)

[0207] reverse primer: GCCGATGGATTGCGAAAT (SEQ ID NO: 6) and the PCR probe was: FAM-AAGAGTTCCTGCAAGAAATGGGAAACATCCA-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:

[0208] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0209] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0210] Probes and primers to mouse PPAR-alpha were designed to hybridize to a mouse PPAR-alpha sequence, using published sequence information (GenBank accession number NM_(—)011144.1, incorporated herein as SEQ ID NO:11). For mouse PPAR-alpha the PCR primers were:

[0211] forward primer: AACGGGTAACCTCGAAGTCTGA (SEQ ID NO:12)

[0212] reverse primer: AGGGATTTAAGAGAGTGCACATAGC (SEQ ID NO: 13) and the PCR probe was: FAM-CGGTCTGTTCCCTTCCTGCCACC-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were:

[0213] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15)

[0214] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0215] Northern Blot Analysis of PPAR-Alpha mRNA Levels

[0216] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0217] To detect human PPAR-alpha, a human PPAR-alpha specific probe was prepared by PCR using the forward primer GGCGATCTAGAGAGCCCGTTA (SEQ ID NO: 5) and the reverse primer GCCGATGGATTGCGAAAT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0218] To detect mouse PPAR-alpha, a mouse PPAR-alpha specific probe was prepared by PCR using the forward primer AACGGGTAACCTCGAAGTCTGA (SEQ ID NO: 12) and the reverse primer AGGGATTTAAGAGAGTGCACATAGC (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0219] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0220] Antisense Inhibition of Human PPAR-Alpha Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0221] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human PPAR-alpha RNA, using published sequences (a genomic sequence of human PPAR-alpha represented by residues 58000-144000 of GenBank accession number NT_(—)011523.7, incorporated herein as SEQ ID NO: 4; GenBank accession number NM_(—)005036.2, representing the main mRNA of human PPAR-alpha, incorporated herein as SEQ ID NO: 18; GenBank accession number BF684348.1, incorporated herein as SEQ ID NO: 19; GenBank accession number BC000052.1, incorporated herein as SEQ ID NO: 20; GenBank accession number AF270490.1, incorporated herein as SEQ ID NO: 21; GenBank accession number BE168040.1, incorporated herein as SEQ ID NO: 22; and GenBank accession number BG259843.1, incorporated herein as SEQ ID NO: 23). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human PPAR-alpha mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which A549 cells were treated oligonucleotides 220833-220910 (SEQ ID NOs: 24-101). The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human PPAR-alpha mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET % SEQ CONTROL ISIS # REGION NO SITE SEQUENCE INHIB ID NO SEQ ID NO 220833 5′UTR 4 1942 cagggccctgagcttcagcc 29 24 1 220834 5′UTR 4 1969 cacaaactgttgagtccaca 72 25 1 220835 5′UTR 4 1999 gacagcttctcagttctgag 68 26 1 220836 5′UTR 18 170 gcgccgagctccaagctctt 79 27 1 220837 Start 4 48424 gtgtccaccatcgcgaccag 69 28 1 Codon 220838 Coding 4 48500 ttgcaggaactcttcagata 85 29 1 220839 Coding 4 48546 tatcctcgccgatggattgc 68 30 1 220840 Coding 4 48559 aagcttccagaactatcctc 81 31 1 220841 Coding 4 48588 ttcctaaatactggtattcc 71 32 1 220842 Coding 4 48612 ccgagccatctgagccagga 70 33 1 220843 Coding 4 48621 ccgtgatgaccgagccatct 64 34 1 220844 Coding 18 410 aaagcgtgtccgtgatgacc 23 35 1 220845 Coding 4 65290 ttcaatgctccactgggaga 47 36 1 220846 Coding 4 65299 cattcgatgttcaatgctcc 0 37 1 220847 Coding 4 65310 cgcagattctacattcgatg 68 38 1 220848 Coding 4 65346 cgtggactccgtaatgatag 40 39 1 220849 Coding 4 68405 cacttgtgaaatcgacaata 58 40 1 220850 Coding 4 68412 agaaaggcacttgtgaaatc 65 41 1 220851 Coding 4 68429 ttgtgtgacatcccgacaga 72 42 1 220852 Coding 4 69865 ttggcattcgtccaaaacga 55 43 1 220853 Coding 4 69876 tttctcagatcttggcattc 39 44 1 220854 Coding 4 69885 cagttttgctttctcagatc 68 45 1 220855 Coding 4 69900 aagaatttctgctttcagtt 63 46 1 220856 Coding 4 69905 caggtaagaatttctgcttt 56 47 1 220857 Coding 4 69910 gttcacaggtaagaatttct 59 48 1 220858 Coding 4 69959 ctcttggccagagatttgag 76 49 1 220859 Coding 4 69979 tcaagtaggcctcgtagatt 61 50 1 220860 Coding 4 70000 ccttgttcatgttgaagttc 50 51 1 220861 Coding 18 914 tgacaaaaggtggattgtta 0 52 1 220862 Coding 4 81895 ccattggccaccagcttggc 43 53 1 220863 Coding 4 81921 ggacctccgcctccttgttc 69 54 1 220864 Coding 4 81991 atggccttggcgaattccgt 33 55 1 220865 Coding 4 82019 gttcaggtccaagtttgcga 43 56 1 220866 Coding 4 82046 tccgtattttagcaatgtca 36 57 1 220867 Coding 4 82057 gcctcataaactccgtattt 45 58 1 220868 Coding 4 82082 cacagaagacagcatggcga 42 59 1 220869 Coding 4 82100 catcccgtctttgttcatca 13 60 1 220870 Coding 4 82119 catttccatacgctaccagc 37 61 1 220871 Coding 4 82164 agaacggtttccttaggctt 49 62 1 220872 Coding 4 82252 gcagccacaaaaagggagat 18 63 1 220873 Coding 4 82262 gcaaatgatagcagccacaa 40 64 1 220874 Coding 4 85181 tttagaaggccaggacgatc 0 65 1 220875 Coding 4 85216 caataccctcctgcattttt 42 66 1 220876 Coding 4 85229 ctgagcacatgtacaatacc 10 67 1 220877 Coding 4 85240 gcaggtggagtctgagcaca 58 68 1 220878 Coding 4 85245 gctctgcaggtggagtctga 29 69 1 220879 Coding 4 85320 ctccgtcaccagctgccgga 42 70 1 220880 Coding 4 85346 ttgatgatctgcaccagctg 7 71 1 220881 Stop 4 85419 tgaaggaactcagtacatgt 40 72 1 Codon 220882 3′UTR 4 85445 aactcctggaaaaggtgtgg 18 73 1 220883 3′UTR 4 85507 ggtggatatttgtgcaaaat 40 74 1 220884 3′UTR 4 85531 ctgtccaagctctaaggtta 25 75 1 220885 3′UTR 4 85558 taatatgccggttacctaca 38 76 1 220886 3′UTR 18 1806 tcccccagcatttgagttct 19 77 1 220887 Intron: 4 1223 gcgcacccacccagggtcgg 23 78 1 exon junction 220888 Intron: 4 2016 ttctatttacctgtggtgac 11 79 1 exon junction 220889 Intron 4 5782 aattctgtgcccaagtttcc 58 80 1 220890 Intron: 4 26881 taaacgtgtatgtacctctt 65 81 1 exon junction 220891 Intron 4 37215 gatgatgcttacagtgttca 31 82 1 220892 Intron 4 37832 caaagaacttgtgaccattt 61 83 1 220893 Intron: 4 38760 gtgtggcactggcacgggaa 43 84 1 exon junction 220894 Intron: 4 38862 gagtacgcacctgagctaat 28 85 1 exon junction 220895 Intron: 4 48381 ctccaagctactgggaggaa 65 86 1 exon junction 220896 Intron: 4 68302 gaaagaagccctgtgagggt 0 87 1 exon junction 220897 Intron 4 71520 atgtcactgtcttttcactg 62 88 1 220898 Exon: 19 88 agcttcagcctgggccgcgg 39 89 1 exon junction 220899 Exon: 19 172 ctccaagctactgtggtgac 58 90 1 exon junction 220900 5′UTR 20 1 tcttgaacttccctcgtgcc 0 91 1 220901 5′UTR 20 71 gggtgtggcactcttatcta 12 92 1 220902 5′UTR 4 38831 acgctggagaccacagacag 53 93 1 220903 5′UTR 20 171 gagctccaagctgagctaat 0 94 1 220904 Coding 4 70096 cgacactggttccatgttgc 50 95 1 220905 3′UTR 4 70191 gaatgaactgtttccatctt 69 96 1 220906 Exon: 21 148 gcttcagcccagggtcggtc 0 97 1 exon junction 220907 5′UTR 4 81789 atgtgggatgcgctatgctc 0 98 1 220908 Intron 4 70322 tggtaagctattaaggtttt 64 99 1 220909 Intron 4 70330 ttagtacttggtaagctatt 70 100 1 220910 Intron 4 70650 tgactcacacctgtaatgcc 37 101 1

[0222] As shown in Table 1, SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 53, 54, 56, 58, 59, 62, 64, 66, 68, 70, 72, 74, 80, 81, 83, 84, 86, 88, 90, 93, 95, 96, 99 and 100 demonstrated at least 40% inhibition of human PPAR-alpha expression in this assay and are therefore preferred. More preferred are SEQ ID NOs: 27, 42 and 49. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is the species in which each of the preferred target segments was found.

Example 16

[0223] Antisense Inhibition of Mouse PPAR-Alpha Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap.

[0224] In accordance with the present invention, a second series of antisense compounds were designed to target different regions of the mouse PPAR-alpha RNA, using published sequences (GenBank accession number NM_(—)011144.1, incorporated herein as SEQ ID NO: 11; a genomic sequence of mouse PPAR-alpha represented by a concatenation of GenBank accession numbers X75287.1-X75294.1, incorporated herein as SEQ ID NO: 102; GenBank accession number AT323000.1, incorporated herein as SEQ ID NO: 103; GenBank accession number BB628277.1, incorporated herein as SEQ ID NO: 104; GenBank accession number BB649343.1, incorporated herein as SEQ ID NO: 105; GenBank accession number BB847654.1, incorporated herein as SEQ ID NO: 106; and a variant of mouse PPAR-alpha represented by a sequence generated from GenBank accession number X75287.1, incorporated herein as SEQ ID NO: 107). The compounds are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the compound binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse PPAR-alpha mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which mouse primary hepatocytes were treated with oligonucleotides 233452-233523 (SEQ ID NOs: 180-275). The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 2 Inhibition of mouse PPAR-alpha mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET TARGET CONTROL SEQ ID SITE % SEQ ID SEQ ID ISIS # REGION NO SEQUENCE INHIB NO NO 233452 5′UTR 11 49 aggcagggccttgaacttca 84 108 1 233453 5′UTR 11 62 cagttcacagggaaggcagg 48 109 1 233454 Start 11 158 gtgtccaccatgttggatgg 74 110 1 Codon 233455 Coding 11 212 ggactttccaggtcatctgc 52 111 1 233456 Coding 11 222 ttcagataagggactttcca 80 112 1 233457 Coding 11 232 gtaagaattcttcagataag 5 113 1 233458 Coding 11 262 gagaaatctcttgaatgttt 36 114 1 233459 Coding 11 356 tctgtgatgacagagccctc 55 115 1 233460 Coding 11 365 gagagggtgtctgtgatgac 26 116 1 233461 Coding 11 463 cacatattcgacactcgatg 72 117 1 233462 Coding 11 473 gccttgtccccacatattcg 84 118 1 233463 Coding 11 665 aagcgaattgcattgtgtga 51 119 1 233464 Coding 11 754 ggtctgcagtttccgaatct 79 120 1 233465 Coding 11 764 agagatttgaggtctgcagt 81 121 1 233466 Coding 11 792 caggtaggcttcgtggattc 83 122 1 233467 Coding 11 826 cccgggccttgaccttgttc 81 123 1 233468 Coding 11 836 gcgagtatgacccgggcctt 81 124 1 233469 Coding 11 868 tgacaaaaggcgggttgttg 50 125 1 233470 Coding 11 880 ccatgtcatgtatgacaaaa 87 126 1 233471 Coding 11 890 cacaaggtctccatgtcatg 83 127 1 233472 Coding 11 965 aagaatcggacctctgcctc 83 128 1 233473 Coding 11 975 gcagcagtggaagaatcgga 75 129 1 233474 Coding 11 985 acatgcactggcagcagtgg 71 130 1 233475 Coding 11 995 gtctccacggacatgcactg 65 131 1 233476 Coding 11 1007 agctccgtgacggtctccac 90 132 1 233477 Coding 11 1036 agcctgggatagccttggca 84 133 1 233478 Coding 11 1046 aagtttgcaaagcctgggat 75 134 1 233479 Coding 11 1094 gcttcatacacaccgtactt 36 135 1 233480 Coding 11 1104 cgtgaagatggcttcataca 65 136 1 233481 Coding 11 1232 gcgaagtcaaacttgggttc 51 137 1 233482 Coding 11 1272 aatgtcactgtcatccagtt 69 138 1 233483 Coding 11 1299 gcaaattatagcagccacaa 74 139 1 233484 Coding 11 1309 gatctccacagcaaattata 64 140 1 233485 Coding 11 1321 gaaggccaggccgatctcca 91 141 1 233486 Coding 11 1331 cctatgtttagaaggccagg 77 142 1 233487 Coding 11 1359 aatcccctcctgcaacttct 66 143 1 233488 Coding 11 1370 agcacgtgcacaatcccctc 90 144 1 233489 Coding 11 1394 tggttgctctgcaggtggag 52 145 1 233490 Coding 11 1476 gagctgcgcatgctccgtga 92 146 1 233491 Coding 11 1501 actcggtcttcttgatgacc 81 147 1 233492 Coding 11 1538 tagatctcttgcaacagtgg 43 148 1 233493 Coding 11 1548 catgtctctgtagatctctt 81 149 1 233494 Stop 11 1555 atcagtacatgtctctgtag 49 150 1 Codon 233495 Stop 11 1562 aggaaagatcagtacatgtc 71 151 1 Codon 233496 3′UTR 11 1630 tccctgctctcctgtatggg 79 152 1 233497 3′UTR 11 1635 gcaaatccctgctctcctgt 90 153 1 233498 3′UTR 11 1640 tctgtgcaaatccctgctct 77 154 1 233499 3′UTR 11 1754 cacccccatttcggtagcag 74 155 1 233500 3′UTR 11 2005 ggccacaccttgacttgtag 83 156 1 233501 Genomic 102 87 gctgcgaacaccaatgttcg 38 157 1 233502 Exon 102 209 ccacgccgtgagaagggagc 16 158 1 233503 Exon 102 270 tctcctctaagttccccgag 27 159 1 233504 Intron 102 358 cttcaacttggcggcagcgt 41 160 1 233505 Exon: 103 205 tttgaaggagctccacagca 37 161 1 exon junction 233506 Exon 104 16 tagcgtgtgccctctccagt 36 162 1 233507 Exon: 104 313 ttcaacttggctctcctcta 29 163 1 exon junction 233508 Exon 105 61 ggctgcactccgcctgcggg 37 164 1 233509 Exon: 105 75 ttcaacttggctgaggctgc 13 165 1 exon junction 233510 Exon 106 76 tctagatcgcacagcttgtt 8 166 1 233511 Exon 106 87 cgttgagctggtctagatcg 0 167 1 233512 Exon: 106 155 ttcaacttggcggccaggac 50 168 1 exon junction 233513 Variant 107 278 gcgcaccggccaggactgaa 83 169 1 233514 Variant 107 510 ggcgagacacaccccctgga 31 170 1 233515 Variant 107 783 ccctgggcacctgaggctgc 83 171 1 233516 Variant 107 926 cctctccagtggctgtgggt 26 172 1 233517 Variant 107 1232 ctccagttacctctcctcta 0 173 1 233518 Variant 107 1277 cagcaaagcctaggctgtga 60 174 1 233519 Variant 107 1880 cagcacttacctgtgatgac 0 175 1 233520 Variant 107 2540 aagcgaattgctggagttgg 23 176 1 233521 Variant 107 3431 ccaggccgatctacgctcaa 84 177 1 233522 Variant 107 3438 tagaaggccaggccgatcta 77 178 1 233523 Variant 107 3547 tttgaaggagctttgggaag 0 179 1

[0225] As shown in Table 2, SEQ ID NOs: 108, 110, 111, 112, 115, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 151, 152, 153, 154, 155, 156, 168, 169, 171, 174, 177 and 178 demonstrated at least 50% inhibition of mouse PPAR-alpha expression in this experiment and are therefore preferred. More preferred are SEQ ID NOs: 141, 144 and 146. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 3. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 3 is the species in which each of the preferred target segments was found. TABLE 3 Sequence and position of preferred target segments identified in PPAR-alpha. TARGET SEQ ID TARGET REV COMP SEQ ID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 137488 18 1969 tgtggactcaacagtttgtg 25 H. sapiens 180 137489 18 1999 ctcagaactgagaagctgtc 26 H. sapiens 181 137490 4 170 aagagcttggagctcggcgc 27 H. sapiens 182 137491 18 48424 ctggtcgcgatggtggacac 28 H. sapiens 183 137492 18 48500 tatctgaagagttcctgcaa 29 H. sapiens 184 137493 18 48546 gcaatccatcggcgaggata 30 H. sapiens 185 137494 18 48559 gaqgatagttctggaagctt 31 H. sapiens 186 137495 18 48588 ggaataccagtatttaggaa 32 H. sapiens 187 137496 18 48612 tcctggctcagatggctcgg 33 H. sapiens 188 137497 18 48621 agatggctcggtcatcacgg 34 H. sapiens 189 137499 18 65290 tctcccagtggagcattgaa 36 H. sapiens 190 137501 18 65310 catcgaatgtagaatctgcg 38 H. sapiens 191 137502 18 65346 ctatcattacggagtccacg 39 H. sapiens 192 137503 18 68405 tattgtcgatttcacaagtg 40 H. sapiens 193 137504 18 68412 gatttcacaagtgcctttct 41 H. sapiens 194 137505 18 68429 tctgtcgggatgtcacacaa 42 H. sapiens 195 137506 18 69865 tcgttttggacgaatgccaa 43 H. sapiens 196 137508 18 69885 gatctgagaaagcaaaactg 45 H. sapiens 197 137509 18 69900 aactgaaagcagaaattctt 46 H. sapiens 198 137510 18 69905 aaagcagaaattcttacctg 47 H. sapiens 199 137511 18 69910 agaaattcttacctgtgaac 48 H. sapiens 200 137512 18 69959 ctcaaatctctggccaagag 49 H. sapiens 201 137513 18 69979 aatctacgaggcctacttga 50 H. sapiens 202 137514 18 70000 gaacttcaacatgaacaagg 51 H. sapiens 203 137516 18 81895 gccaagctggtggccaatgg 53 H. sapiens 204 137517 18 81921 gaacaaggaggcggaggtcc 54 H. sapiens 205 137519 18 82019 tcgcaaacttggacctgaac 56 H. sapiens 206 137521 18 82057 aaatacggagtttatgaggc 58 H. sapiens 207 137522 18 82082 tcgccatgctgtcttctgtg 59 H. sapiens 208 137525 18 82164 aagcctaaggaaaccgttct 62 H. sapiens 209 137527 18 82262 ttgtggctqctatcatttgc 64 H. sapiens 210 137529 18 85216 aaaaatgcaggagggtattg 66 H. sapiens 211 137531 18 85240 tgtqctcagactccacctgc 68 H. sapiens 212 137533 18 85320 tccggcagctggtgacggag 70 H. sapiens 213 137535 18 85419 acatgtactgagttccttca 72 H. sapiens 214 137537 18 85507 attttgcacaaatatccacc 74 H. sapiens 215 137543 18 5782 g gaaacttgggcacagaatt 80 H. sapiens 216 137544 18 26881 aagaggtacatacacgttta 81 H. sapiens 217 137546 18 37832 aaatggtcacaagttctttg 83 H. sapiens 218 137547 18 38760 ttcccgtgccagtgccacac 84 H. sapiens 219 137549 18 48381 ttcctcccagtagcttggag 86 H. sapiens 220 137551 18 71520 cagtgaaaagacagtgacat 88 H. sapiens 221 137553 19 172 gt caccacagtagcttggag 90 H. sapiens 222 137556 18 38831 ctgtctgtggtctccagcgt 93 H. sapiens 223 137558 18 70096 gcaacatggaaccagtgtcg 95 H. sapiens 224 137559 18 70191 aagatggaaacagttcattc 96 H. sapiens 225 137562 18 70322 aaaaccttaatagcttacca 99 H. sapiens 226 137563 18 70330 aatagcttaccaagtactaa 100 H. sapiens 227 149975 11 49 tgaagttcaaggccctgcct 108 M. musculus 228 149977 11 158 ccatccaacatggtggacac 110 M. musculus 229 149978 11 212 gcagatgacctggaaagtcc 111 M. musculus 230 149979 11 222 tggaaagtcccttatctgaa 112 M. musculus 231 149982 11 356 gagggctctgtcatcacaga 115 M. musculus 232 149984 11 463 catcgagtgtcgaatatgtg 117 M. musculus 233 149985 11 473 cgaatatgtggggacaaggc 118 M. musculus 234 149986 11 665 tcacacaatgcaattcgctt 119 M. musculus 235 149987 11 754 agattcggaaactgcagacc 120 M. musculus 236 149988 11 764 actgcagacctcaaatctct 121 M. musculus 237 149989 11 792 gaatccacgaagcctacctg 122 M. musculus 238 149990 11 826 gaacaaggtcaaggcccggg 123 M. musculus 239 149991 11 836 aaggcccgggtcatactcgc 124 M. musculus 240 149992 11 868 caacaacccgccttttgtca 125 M. musculus 241 149993 11 880 ttttgtcatacatgacatgg 126 M. musculus 242 149994 11 890 catgacatggagaccttqtg 127 M. musculus 243 149995 11 965 gaggcagaggtccgattctt 128 M. musculus 244 149996 11 975 tccgattcttccactgctgc 129 M. musculus 245 149997 11 985 ccactgctgccagtgcatgt 130 M. musculus 246 149998 11 995 cagtgcatgtccgtggagac 131 M. musculus 247 149999 11 1007 gtggagaccgtcacggagct 132 M. musculus 248 150000 11 1036 tgccaaggctatcccaggct 133 M. musculus 249 150001 11 1046 atcccaggctttgcaaactt 134 M. musculus 250 150003 11 1104 tgtatgaagccatcttcacg 136 M. musculus 251 150004 11 1232 gaacccaagtttgacttcgc 137 M. musculus 252 150005 11 1272 aactggatgacagtgacatt 138 M. musculus 253 150006 11 1299 ttgtggctgctataatttgc 139 M. musculus 254 150007 11 1309 tataatttgctgtggagatc 140 M. musculus 255 150008 11 1321 tggagatcggcctggccttc 141 M. musculus 256 150009 11 1331 cctggccttctaaacatagg 142 M. musculus 257 150010 11 1359 agaagttgcaggaggggatt 143 M. musculus 258 150011 11 1370 gaggggattgtgcacgtgct 144 M. musculus 259 150012 11 1394 ctccacctgcagagcaacca 145 M. musculus 260 150013 11 1476 tcacggagcatgcgcagctc 146 M. musculus 261 150014 11 1501 ggtcatcaagaagaccgagt 147 M. musculus 262 150016 11 1548 aagagatctacagagacatg 149 M. musculus 263 150018 11 1562 qacatgtactgatctttcct 151 M. musculus 264 150019 11 1630 cccatacaggagagcaggga 152 M. musculus 265 150020 11 1635 acaggagagcagggatttgc 153 M. musculus 266 150021 11 1640 agagcagggatttgcacaga 154 M. musculus 267 150022 11 1754 ctgctaccgaaatgggggtg 155 M. musculus 268 150023 11 2005 ctacaagtcaaggtgtggcc 156 M. musculus 269 150035 106 155 gtcctggccgccaagttgaa 168 M. musculus 270 150036 107 278 ttcagtcctggccggtgcgc 169 M. musculus 271 150038 107 783 gcagcctcaggtgcccaggg 171 M. musculus 272 150041 107 1277 tcacagcctaggctttgctg 174 M. musculus 273 150044 107 3431 ttgagcgtagatcggcctgg 177 M. musculus 274 150045 107 3438 tagatcggcctggccttcta 178 M. musculus 275

[0226] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of PPAR-alpha.

[0227] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 17

[0228] Western Blot Analysis of PPAR-Alpha Protein Levels

[0229] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to PPAR-alpha is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 18

[0230] Targeting of Individual Oligonucleotides to Specific Variants of PPAR-Alpha

[0231] It is advantageous to selectively inhibit the expression of one or more variants of PPAR-alpha. Consequently, in one embodiment of the present invention are oligonucleotides that selectively target, hybridize to, and specifically inhibit one or more, but fewer than all of the variants of PPAR-alpha. A summary of the target sites of the variants is shown in Table 4 and includes GenBank accession number NM_(—)005036.1, representing PPAR-alpha main mRNA (represented in Table 4 as PPAR-alpha), incorporated herein as SEQ ID NO: 18; and a sequence representing the truncated PPAR-alpha variant (PPAR-alpha-tr), incorporated herein as SEQ ID NO: 276. TABLE 4 Targeting of individual oligonucleotides to specific variants of PPAR-alpha OLIGO SEQ ID VARIANT SEQ ISIS # NO. TARGET SITE VARIANT ID NO. 220836 27 170 PPAR-alpha-tr 276 220851 42 703 PPAR-alpha 18 220852 43 729 PPAR-alpha 18 220853 44 740 PPAR-alpha 18 220854 45 749 PPAR-alpha 18 220855 46 764 PPAR-alpha 18 220856 47 769 PPAR-alpha 18 220857 48 774 PPAR-alpha 18 220858 49 823 PPAR-alpha 18 220859 50 843 PPAR-alpha 18 220860 51 864 PPAR-alpha 18 220861 52 918 PPAR-alpha 18 220863 54 800 PPAR-alpha-tr 276

[0232]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 276 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 3 atgcattctg cccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 86001 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <400> SEQUENCE: 4 gacaaggtcc ctcccgggcc gcctcccacc ctacgcactt ctgagcctca agggcacccg 60 gtcccgggtc ccgggtctag accggctcat cgcacagagt agcagagccg ggctcatcga 120 ggaggcagga ggggctcgcc agcgtggcac gggcgcccgg cgggaacctc cacccgcccc 180 gcggccgcgc gtccccgcct cgaattcagc cccgccccgg tgcgccgggc tggaggggcg 240 ctgacgctca gcggtgtccc atcggtgacc ttggacggtc cctccacctc tccggcctca 300 gtttcccttg gctgcagcgg ccgcggggcg ctaggtggga gccgctgagc gctcccgggg 360 ccccgcccac cgcgagcagc caatcgggcg ccgccctccg gggggtgtgt cccggggccg 420 aggcccgggg cccggagggc gcgcggggcg ggcggggctt ccgggtcggg cctcgggaca 480 ctggctcgcg cggaccgggg cagggggcgg gccgaggggc ggtgcgtgtc gcgggggcgc 540 ggctggcacg gacgcgcgga ggcggcgccg ggcatgggcc gtggacgcgg cggccccgcg 600 gcgggggcag cgggcggcgg gggcggaggc ggccgctagc gccctgcccg gcgccgcctc 660 cttcggcgtt cgccccacgg accggcaggc ggcggaccgc ggcccaggtg cccgggggcg 720 ggcgggcggg cgggcgggaa cgcgcgcggg ggtccgcggt ccgggcttcc caggtcccgg 780 gacccggagg gcggcggacg ggggaggggc aggggctggg cggcgcatgc gcggggcccg 840 gggtctcggg gtctccgggt cccggggacc cgggggcccg gggtgcgcgg ctggggacct 900 gagggcgagg agcgaggaca cacaccgagg actcttgcga gggatctcgg ggcccagctc 960 ggcctccctc ctagcgctgg gggcctgccc ggaacccgag tccgcggctg tccctggggt 1020 ttggcgctgc gcggaggtcg ggtctgggga ccgcagcgac tctgggtctt cgggttgtcc 1080 cctcggaggg agggcccacg ggcggggaca tcgggacttg ccctttcctc ggcgcagcgg 1140 agctggggcg tcgccgactc agaaggtgct ttccgagacc tccagggatc tccgaggcga 1200 ggaaacccgg gccccggaca gaccgaccct gggtgggtgc gcccggcttc tgccgtcgga 1260 cggagacgcg cgtgtttgtt cctccagctg cgaccacctt tgaggaacgg ttcccacttt 1320 gtgccccaac gcggcggggc gaccccggac aggctgcgct gggccgggtg gcttctctgc 1380 ggaagccgcg ccacgtcgct cccggtcggg gccgctgagg gtcgggcgcc caggtctttc 1440 cggagtcccg ggctgcgcgg cccgcgtggt gcgggtgaag ctggaggggc gcggggtggt 1500 gccagtggaa gtcaggaggg tcggccctgc cccctcacgc accccaaccg ggcacaactg 1560 cacgcctgtg cttttctgaa gtctttttta aaagttaaaa gagaggaagt gtgctccaag 1620 tgtcaggatt ctttccaaga aaaacccaca gttgtccaat ggcctgggct tcgtgggacc 1680 tccggggctg cacgcccacg tcagcctcag ccgacccctg ccaggaaacc agggaggccc 1740 ctcctctccc agcctccttg ggataagggt gccttgggga actgggtcag ggcaaggaca 1800 cgggattttc ctgggaagga ccctgcgaca cccgtgtcgt tgcggggcag ggtcagcatg 1860 actttcctct tccaaggtga agagttgggg ggcatccaga gaacaaccgt aatcacttcc 1920 tccttcacct tcttactgcc aggctgaagc tcagggccct gtctgctctg tggactcaac 1980 agtttgtggc aagacaagct cagaactgag aagctgtcac cacaggtaaa tagaaggttt 2040 aatttactgt ttccagatgg aaatatttaa gtgttttcag tgtttacttc tgttgcacta 2100 cagaccagca atctgggggt tattactttg tgatgcaagg ttagatacgt tttcagactg 2160 aaagtaaaat acatgtgcat ggattcattt tttttttttt tttttttttt tgagacggag 2220 tctcgctctg ccgcccaggc tggagtgcag tggcctaatc tcagatcaca gcaacctctg 2280 ccactggggt tcaagcgatt ctcttgcctc agcctcccga gtagctggga ttacaggcgc 2340 ctgccaccat gcccagctaa tttttgtagt tttagtagag gcggggtttc accatcttgg 2400 ccaggctgat cttgaactcc tgacctcatg atccacctgt tcctcccaaa gtgctgggat 2460 tacagacgtg agccaccgtg cctggcctag gattcacttt gaagttctga gttattgtgt 2520 gacttttgct aggaacttca ttgcttcgtg gcaggcatgt tttgtataat ttaaaacttg 2580 atgacattaa ctttgagaaa cgtgagtgct tactagaccc ttgggatgtc cacactgact 2640 ggtaccgagt agtgtactgt ctctgagctg ttttcatttt gatttgaata ttaagcagat 2700 ggcttcttga gatagacccg tgccagaaca tgccagggat aggctgaaga aacgggccag 2760 atgatacaaa tttgtgtggt caccatccat gagagaccag ggacactggg gctgatgatg 2820 acctctgcaa ctctgaagca aaagtaaact aattggcaag ttgggtgcgg tggctcactc 2880 ctgtaatccc agcactttgg aagctggggt gggcagatcg cttgaggcca ggagttcgag 2940 accagcctgg ccaacatggt gaaaccttgt ctctacaaaa aaatagaaat attgcctggg 3000 catggtggcg gacatctgta atcccagcta ctcaagaaac tgaggcagga gaatcgcttg 3060 agcctgggag gtgaaggttt tagtgaactg agattgtgcc actgcactgc agcctgggcg 3120 ccagggcgag actccgtctc aaaaataaat aaataaaata aaattaatta actaattgac 3180 attagaaaaa aatgtttttt ctttcttttc ccacatcctt tttttttttt tttttttttt 3240 tgtgacagag ttttgctctt gtcacccagg ctggagtgca gtggcatgat cttggctcac 3300 cgcaacgtcc acctcacgga ttcgaacaat actcctgcct cagcctcccg agtagctggg 3360 attacaggca ctcaccacca cacccggcta atttttgtat ttttagtaga ggtgggtttc 3420 accatgttgg ctgggctggt ctcaaactcc tgacctcagg tgaaccgcct gccttggcct 3480 cccaaagggc tgaggttaca ggtgcgagcc accgcgccgg gcccttttcc gacatcttaa 3540 acgtaaagta ggagacgtgt cataatcatc gaatactgca gtggttttca ttagctcctg 3600 tttgtcaaac ttatgaacag agttttaaaa attgtgtatc agccgggtgc ggtggctcac 3660 acctgtaatc tttgggaggc tgaggtgggc agatgacaag atcaggagtt tgagaccagc 3720 ctggccaata tggtgaaacc ctgtctctac taaaaataca aaaattagct gggcatggtg 3780 gcgggtgcct atggtcccag ctactcagga ggctgaagca ggagaatctc ttgaacccgg 3840 gaggtggagg ttgcagtgag ctgagatggc accacagcac cccagcctgg gtgacagagc 3900 aagactccgt ttccaaaaaa aaaaaattgt atatgagaga gacagaacta gacagagaag 3960 aaggagaaaa tgtgtcttct ttatacacta ttttgtaact tgctttatcg agtaggttat 4020 gaaaaatctt cctatgtgaa aaacatttct gcatcatttg aaatgtctat ataatatccc 4080 attgtgttta gatacaataa tatttagcca atctctttat gtgtatatat ttaatacagt 4140 cattctataa atattgactg agtagctgct gtgggctact gtccgcagtg ctgaacaaga 4200 caagcatgaa tccatgaaac tgattttcat accagaatat aaaaaagaaa cttaaagata 4260 atcctcatca tggtaaaaga tgaagaacct atttttgccg ggacatctta ctctttagta 4320 attggtggcc agtgttcttt ttcttgcatg ctgttttgga gagtctgttt tttaaataaa 4380 tatttaagta gcctgggcgc agtggctcac gcctatggtt tcagcacttt gtgaggccga 4440 aggggatgga ttgcttgagc ccagggcttc aagaccagcc tgggcaacct ggcgaaaccc 4500 tgcatctact aaaaatacaa aaattagcca ggtatagtgg cgtgtgcctg tggccccatc 4560 tacttgggag gctgaggtgg gaggatccct tgagcctgag aagtggaggt tgcagtgact 4620 gagatggcac cactacactc cagcctgggt gacagagtga gacctggtct caaaaaataa 4680 ataaatattt atgtaatcat ctttaagcag tgtttttaat tttatttatt tatttattta 4740 tttttgagac agggtctcac tgtgtcacct aggctagagc acagctgcat gatcacggcc 4800 tattgcagcc tcgacctccc tgggctcagg tgatcctccc acctcagcct cccaagcagc 4860 taggaccaca ggcacacgcc accaggcctg actcattttt gtattttttg cagagacggg 4920 gtcttgctat gttgttcaga cctgtctcaa actcctgggc tcaagccatc ctcctgcctc 4980 ggcctcccat agtgctggga ctaagccatg aaccactgca cccggcataa gtggtctttc 5040 tttaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaccacat taattaaaat atgtatttgc 5100 ttattataaa tatatttgaa acatgccaat ttttcttctc tttttttgct ctattggttt 5160 ctgtgtgtgg atggatatat ttttaatggc aaataggatg agtgtcttta cttccaagta 5220 gtcagtgttt ttctttaatg tttgtactaa ttttgtcaca ttgcagttag aggttgtggc 5280 ctgtctaatt tctgcttttt tggaacttga gagtctctgt ttttatttgt ttttggtagc 5340 ctggcataga gtccattttt cttttctttt ctttttttga gacggagttt agctcttgtt 5400 gcccagactg gcgtgcagtg gcgcaatctc agctcactgc aacctccgcc tcctgggttc 5460 aagcgattct cctgcctcag tctccccagt agctgggatt acaggtgccc accaccacac 5520 ctggctattt tttgtatttt tagtagagac ggggttttgc catgttggcc aggctggtct 5580 cgaactcctg atctcaggtg atccacccgc ctcggcctcc caaagtgctg ggattacagg 5640 tgtgagccac tgcgcccagc tgtagatact ttttaaaaag gtatagtttc tgattatggg 5700 gtagaaatgt gctatgtctg tcatttcagc cttatgaatt gcccagaata agctagatca 5760 cctttaaggc catgtggtta gggaaacttg ggcacagaat ttacattttc aacttggtga 5820 taagatgggt ttaaggtaag aatcaaatag gagaaagcct tagctgttcc agcggcccat 5880 gtttaaaaga atgtgcttct ttttccaagt atttctgccg cttgcatgca ctgagcttct 5940 ttggaaagga gcaccatgca ggcatatttt ccagacagga ccggatttgc tcgttactca 6000 gaggtgtgtg cattctttgc ttttaggata tttaattagc atcttttaat agtgatatta 6060 cggtgtctta aaagtttatg catttgaaaa gaaaagaact tactccttgc caggtctcaa 6120 cctatcatgg ttatctttgc agctgagctg cgttggtttt gaggctcaca tatggtaaaa 6180 gtggttggaa atctggaaat attgctgtgt atctgcaaag cagcttgata tagtggaaaa 6240 ggtattaggt cattaatcat gagatttgga ttctagcccc ttagctgctg cctgccaggc 6300 ctggagacct ttgttctctt ctttaaactg ctgctttctc atcagaaaat gaagttcctc 6360 tccataccac ctctctgaag ggctgtgaag ctcgaagtgg cagcttaaaa aactgcccat 6420 ctcaggaggt gtcttaagaa ggaggacata ccgctggctc ctgcctttct cacttagcca 6480 ggtctgatac ctgtgttgtt ttcactgtgg ccattttagg atttttcaaa ggctttcaga 6540 aagcaacatg ctaccgtacc ccttatacac caaaactggt tttcattttg gaatataaaa 6600 gtgagatttc tccaccagta caataaagtt gttacaagtg gttcctatgt gtttgttttt 6660 gtttttgaga cagagtctca ctctgtcacc caggctgcag tgcagtggca caatcttggc 6720 tcactgcaac ctccgcctcc cgggttcaag caattctccc acctcagcct cctaagtagc 6780 tgggactaca ggcacccgcc accacgccca gctaattttt gtatttttag tagagatgga 6840 gtttcaccat gttggccagg ctggttttga tcttctgacc tcaggtgatc cacccgcttc 6900 agcctcccaa agtcttagga ttacaggcgt gagccaccac acccggcctc ctgtgtgttt 6960 tgaaggcgat tgtgacctca ggttttggca gggctatacc ttgtgtttgc tcttactcca 7020 actccatggc atacctggac caggcctctt catcttgaag agggatctgc tgaaatgcag 7080 gcccagtgaa tctccccatg cctggacaca gttccgtcaa gccaggaccc ggtgctgcct 7140 gcacccctgt ttctgttagt ctgactgtcc tcactgagtc taactccttg agggcagaga 7200 ggatgtctta tttatttctg ccccgctagc cgtgtaaact gagtaggtac ttgtaaatgt 7260 tcattgaata agtacctgat taatagaatt taattcaaga agaatgtatt gatgggcctg 7320 tgtggtcacc acagtactga gatgtaggtg ggagctggct gaagggggag gcacctaaac 7380 aggagcgcag acagcggcac ctacggatga tggcccgctc catcccaccg cagcgaaatt 7440 gtcccagacc tctgcagctc cccccacacc tagactgaga gagagctctt cttccttctg 7500 tagggagcag gtgtttcctc cagatgtcca atatgtacct cccattacag cggtgttagg 7560 aaggtgaggg ctgccgctga aagggtcccc ttcataatca tcactagatt tggggtatat 7620 tatggattaa atagaatttt tataagatga cctggggatc tatttaaata aaatcctctt 7680 tctttctgca agatcatgga tttaaattca acacaactga cttcataggg aaggggtatg 7740 gtgaaaggga agtgaggtgg gcagcactga tatttaacaa ggtgagggtc cttctcctgc 7800 tgtgactgtc acattaaaat attcccagga gaaattggag aaaactcaga tgaaatatcg 7860 tctgtgttcc aggaggcagg actcatcgga atgcttttat tttgctccat tttaagagat 7920 ttgcagataa agaggagtga agatttctat tcagatttac ttgctttata cttttaactt 7980 atagaccaca agccaacttt cgaaagagca tcattttgaa tagtaagagt taggaaggca 8040 aatacagaag gactaatggc ttccaagatt atgagcttca taggaatggt ttgagatgag 8100 gctatagtaa agcagaatat tgaagttccc ccaccccctt tcatttttca tttttcattt 8160 ttaagagtga gcgaggccag gcgtggaggc tcatacctgt aatcccagca ctttgggagg 8220 ccgaggtggg cagatcacaa ggtcaggagt ttgagaccag cctggccatc atggtgaaac 8280 cctgtctcta ctaaatgtac aaaaattagc caggcttggt atcaggtgcc tgtaatccca 8340 gctactcagg aggctgaggc aggaaaattg cttgaaccca ggagtcggag gttgcagtga 8400 gctgagatcg caccactgca catctcagaa aaaaaaagag tgaggcccca agtttttttg 8460 catttgtttg taactgaata cgtctgaagt tatgtgataa ccacgccaag gtgacaaatt 8520 gccaagtttc agtaaaagag acccagttat ttagaggttg acacgtggat atgtcccttt 8580 ctaagaagtt cgtggtcagc tttacatgag tatttaaatg cgtgtttata attcagcaat 8640 atggcttgta aaatacagat tgccaatcaa gtgacatgca aatcttgatg atctgaaaca 8700 agttttcttc tgttatctat ggaagaaatg gtaataggga tatttaagtg ggatgaattt 8760 tttgaagcat tttcaggcag ttttccacat ggaacaaaat aacattgagt gggctgctaa 8820 catgaggaac atattgccct ctgcctagga ttatgagtaa atttgataaa ttctagactg 8880 cagtctcatt ttagctcatt ttatgaggca gcttgacaac tgggatagtg tctctttttt 8940 ttgtcggggg tgttgaggct ggagtctcgc tctgctgccc aggctggagt gcactggcgt 9000 gatctcggct cactgcaacc tctgcctccg gggttccagt ggttctccta cctcagcctc 9060 ctgagaagct gggattgtag gcatgtgcca ccacgcccgg ctaatttttg tattttttag 9120 tggagacggg ctttcaccat gttggccagg ctgggtctca aactcctgac ctgaagtgat 9180 ctgcccgcct cagccaccct aagtactggg attacaggca tgagccacca cacctggctt 9240 ctgtttttct atctgtgcat tggggatgaa attaacacaa atgatgttta aagaaaaaaa 9300 tgctcagaga agttagaaat gtgctttaaa ttggaatcat ctcttagtat gtaaaagttt 9360 tttgtaatag aaacaagcag ggcagtattt gacctgttga cagtgtcctt ggactttaca 9420 atttgtgaag cagcgtattt tgcttgagtt gtacgattgt cgtttttttc cctccacttt 9480 gacaactgtt acagaacctg tcaccagata caggcaaggg aggttgggct tcccatctct 9540 gcacggcttc cctgtgattc acaagcaagc aatcagaagt gcacaaaagt ttagaacgcg 9600 attttcattc tcttctttcc ttagaaaaac tcgctttgtt agccttttcc agaaaggaag 9660 gcactcaatt gttgtaatac tcaaatcata aaaagaagcc tagtctagtc tattcagcaa 9720 ggtgttctga aagagggaat tttttaagtt caattatgcg aagatcttga aggtgggact 9780 caaaggagag ggctatcctg ggaagaaggc tttggaaaat gagaggcatg aaggggagag 9840 ggtatttaaa tgtgtttgaa gccaaggatc cttgagagaa aaagctggca ctaacagcgt 9900 tcaaagaact tgcgtgacaa gtgatgacta atgacactga gggtgggttg tgggtgccta 9960 gtgaattcct ccgaagccaa gagagaggtt tccagaccca gggaagaagg tgtgtacacc 10020 cagaagtagt gtagggacag agattccgat cacaagctgt gactggaaga cgccgaccac 10080 cactgcagca gcctgaaaac cacagtcttg aaccgccagc gaagggctgg gaagtgcgga 10140 tccagggctg gtgcactgaa cccagaggag caggctccca ttcccagcca agggtggcag 10200 ctggcgggga tctttccagc agaaagctgt aagtggaagc tttcaattca gagcagtagc 10260 aatgccttca aagtcccagg cttcacgtgg gaacagagaa tgtgaagagt atttagcagg 10320 atgccaatat aagaaatcta tattggtgtt cgtttgtttg tttttgagat ggagtctcgc 10380 tctgtcaccc aggccggagt gcagtggtgc gatctcagct cactgcaatc tctgcctcct 10440 gggttcaagc gattctcctg cctcagcctc ctacatagct ggtactacag gcacgcgcca 10500 ccatgcctgg ctaaattttt gtatttttag tagagatggg gtttcaccac gttggccagg 10560 ctggtctcaa actcccggcc tcatgatccg ccctctgcag cctcccaaag tgctgggatt 10620 acaggcgtga gccaccgcac ctggcccaat attgtttgtt tatttatttc ttgacaggat 10680 ctcactctgt caccaggctg gagtgcagtg gtgtgatctc agctcactgc aacctccacc 10740 tctctggctc aagcaatcct cccacctcag cctcctgagc agctgggact acaggtgcac 10800 accaccacac ccaactagat tttgtgtttt ttgtagagat ggggtttagc catgttcagc 10860 tagtctcaaa ctcctgggct caagtgatct gtccgccttg gcctcccaaa gtgttgggat 10920 tacaggtgtg attcatgatg tccagcccag tatttttctt tcactctgga aaccaaaaat 10980 tattggcttt ttttcctgtt gcattccctt tacttagatg aatctagcaa ggttggctgt 11040 tagtgtctag gtcagaagtc taagtgaaag tgaatattta accacactca agcacagctg 11100 atgatcttta atactaatag aggtataaga cttaaaagaa acaagaaccc agagggaaaa 11160 tatggccatg gactcagaga aaaccacggc agcttccatg gactcataaa aagagctcaa 11220 aacctaggaa gtggatggag actctttttg gaatgaatga attcaaatgt gggctttctt 11280 agtagattaa atcattttct agaaggaatt tcggaaggat gtgtgcccaa ttatggtatc 11340 aggtctgttg tagactcttc aaggaggaag cctctgaaag acaagaagga acaattaaaa 11400 attagaattc aggtgagtgg atcacgaggt caagagatcg agaccagcct ggccaacatg 11460 gtgaaacccc gtctctacta aaaatacaaa atttagctgg gcatggtatg gctgtagtcc 11520 cagctactcg ggaggctgag gcaggagaat cacttgaacc cgggaggcgg aggctgcagt 11580 gaaccaagat tgtgccactg cactccagcc tggcaacagc gagactccat ctcaaaaata 11640 ataagtaaat aaataaataa ataaaaatta gaactcagaa aaggaattaa tttcttctga 11700 gagagaaaaa gatgagattc tagcctaagg tgtaacacat ccatccacca ggtatcattt 11760 ttatacacgt gaagttaaat caccaaagga ccaggtgagc agatgtggac tttccgactg 11820 tgtgtgtgcg acttcctcag agccctcagt ggcgttccct tttccgcgct agcgtttggt 11880 ccctgcgctt ttctggatgc ccccaccccc tctggctcca cgaggccccc tgtacgtcac 11940 catcaccttt gtgagcttga aacctgtcac ccacccgcct tccagatgtc acctgggccc 12000 tcccggaggc cctcgccctc agtgtgtctg attctgagct gtcctgcgtt ttcccctccc 12060 ctcaccctgg cgaccctttt cggtctcagt tgccagcctc ctgctagggc tgggtggggg 12120 acatcaaagg caggacaagg tgtagggtcc tcacccacca cttagcagct ctcagatgca 12180 gacagatttt tcagctggcc tgtggctcag tttccctcag ctacaagagg ggtgcatgct 12240 agggtttctc tggattgctg cacctggcag gtagtgtgag cttggtaggt gcttccttgc 12300 ttatcattgc ctctcccatc ttaatgttgt cccatccatc caatgtttat gggatgagag 12360 gttgatagga gggcatggcc ctgacattcc agggactgac cgacacgctg tctacacaaa 12420 ccccttctgg ttcttctcgt gcactgggcg tgccggagac acactccctt accctcatac 12480 cccgccgcac ccctgtgacc tctacctttg agacctcagc ttaaactcac tcttagggaa 12540 ggctccctga accacctgct ggggttgtat gctgatgcag gtacttgtaa cacctggtgc 12600 ttttcctttc gtgcactcag gcagtgttta ttcaagtgat ggcttggtga cagtggctct 12660 ccctgagacc ccgtgaggcc agagtccttg gctcatcact catggttgaa cccggagcct 12720 cttgctggta ggtgcttcag gactggctct gggagcctgt ggctcctgcc gggtacccac 12780 cggttgagat acctcaagtt ttaaatgcca cctccttcct gaagccttcc ttgctgctcc 12840 cccaaactag aggcaggagt tttgtccttc agataaccta tggcatttga gtcactctga 12900 tttgatgaat tctgccttca cttgagcagc tattaggggc atatgtcagt cattcattcc 12960 tcagttcatg tatttattca gcaaatattt actgagcacg tactgcgtgc caggcactgt 13020 cctgctgtgg aaaacagcag gcatgattcc ctgccactac caaccactgc atcgcataac 13080 tggcagactc ccagcttcaa ggagaggcac ggagggaaac tgagagcagc ctgcagaggg 13140 gaaagagcgg ggacagaggg tcacggaggt cgcaggggcg tgtgtgcagc acctgccagt 13200 gaacggaatg tgcggctcca gatgtcgttg tctttaaact tcggaatttc ctttcactaa 13260 agaaccaagt ccagggggag gaaagagtga atacaaatta tccaagaaac tcaagagctc 13320 attttagttc tcctgattat gatcttaaag gcattaagcg ctcaagttaa actccttgtg 13380 acccacatag gttagcagaa tttaaatcct aggtgattct taactctaat catacatcta 13440 atgacctata ttgaagatac actgcctgct tagttgtggc ttcagccttt gctccgtcac 13500 tgatagttct agcctgaaaa gcaaatgagc cctcatgctc acgatttcac cacagtcaca 13560 taagcgggaa gagcaggctc ctggctgtgg cgagcttgac tccatttggt ttgatagaaa 13620 tgagaggtag atgattccct agacaaatgc aggcctttct cgaagcccct ttcccaggac 13680 gacgtgacat gagtggtctg tgccttccag ggcagccacg tcatgctttg cccagccagg 13740 gcggtgggga gggagacagc cacatcctgc ccggggctcc tgggccccgc tgcatcaagt 13800 gaaagcaggg ctggctccct gatgtccttg gagaagtcgc ccacactgct ttcccccatg 13860 ggagtgacaa ggatgtgtcc cgccagcctt ccacgacgga ccccccactc tctattaatt 13920 cccaagaaac caggccatgg aggtgggttt gagggtttgt attggtgttt tttaaagtca 13980 ggttgaccga gtgcggtggc tcacgcttgt aatcccagca ctttgggagg ctgaggcggg 14040 cggatcacat gaggtcagga gttcaagacc agcctggcca acatggtgaa accttgtctc 14100 tactaaaact acaaaaaaaa ttacctgggc gtggtggtgg gcgcctgtaa tcccagctac 14160 tcaggaggct gaggcaggag aaacccttga actagggagg ctgcagtgag ccgagatcgc 14220 gccactccag cctgggtgac aagagtgaga ttctgcctca aaataaataa agtcgggttt 14280 attaagatat aatttacata cagtaatttt tttttttttt gagacagagt ttcactcttg 14340 ttgcccaggc tggagtgcaa tggcacgatc tcagctcact gcaacctccg cttccagggt 14400 tcaagccatt ctactgcctc agcctcctga gtagctgaga ttacaggtgt ccaccaccat 14460 gccttgctaa tttttgtatt tttagtagag acaggttttc gctatgttgg ccaagctggt 14520 cttgaactcc tgacctcagg tgatccgcca gcctcggcct cccaaagtgc tgggattaca 14580 ggcatgagcc actgcacccg gccagtacat gctttcttga tttgtctgtt tcccacctgt 14640 ctcccctccc tagaatggca gctccatgac gacagaggtg tttctctgtt ttctccatgg 14700 ctgcaccctc agctgctaga aggtggccca gcataggagg tatttaatga agccttcctc 14760 tccacttaaa tctacaccct tgtgcttatt aaaaggtgac agttttctgt ttgaaaattt 14820 tattagtgtt ttaatgagaa agttattatt tgggtaatgc ctgaatatga ggaaaacatt 14880 aagggtagaa atgtaattgt tttcctattt cattcagtct atggatttta ttgaagatta 14940 cagaattact tctttgtagc tatggaagta aaaaaataat aagacgagta gctatttcaa 15000 aacgtagggc tgataaattt gggatggttt gagaacgtta agttggggaa ctccatttct 15060 ttttttacat ttttatttat tttcatttgt ttatttattt atttgagaca gagtttcgct 15120 ctgttgccca ggctagagtg caatgccatg atctcggctc actgcaacct ctgcctccgg 15180 ggtataagtg attctcccat atcagcctcc cgaggagctg ggactacagg cgcctgccac 15240 cacacctggc taatttttgt atttttagta gagatgggat ttggccatgt cagccaggct 15300 ggcctcaaac tcctgaccgc aggtgatccg cctgcccttg gcctcccaaa gtgctgggat 15360 tacaggtgtg agccaccgcg cccagccagg gaactgcatt tctgacagtg gctcagtagt 15420 ttggaagtta actggcaaag gtggacagaa tctttaaaca tatgtggagg aattggagag 15480 tttacaagat agtgaagaac tgccaggcca tggtctggag aagatggaaa cttgatgttt 15540 ggggccattg tgtccctggg gtgttggcca atttatgaaa gaagcagtta agagcctgag 15600 tggcactttt gaggggctag aagggaagac cctggtaaac atcccaaact ttggattggg 15660 acccaaaaaa gctccatccc aggagtacag gtgacctgga aacggatcag cgtaatcgag 15720 gactgaagtc cagttctagc tacgcccagt ccttgagact ggattaaggt gatctcagat 15780 tgcaaggacc tcaaatgcct ggcagaagca agtgaatatc cttctggagg aacagagcct 15840 catcctaggc ctctaattat ttttaaggac aatttttcaa atgcaggctt tcctcccttt 15900 gcacagttcc cttatgcata aatttcagtc agtggccagc tgcagtggct catgcatgta 15960 atcccagcgc tttgggaggc caaggcgggt gaattgcttg agtctgggag ttggagaccg 16020 gcctgggcaa catagaaccc catctctatt tttaaaaata aaatattaat tatcactgct 16080 tagttaaatt atagtggtct cccaacaata cagatcagat cccagctccc atggtatata 16140 cactgtgagt gctgtataaa gtacaagctc tgccgccagt tctccagcct acaaatcaca 16200 gtatagataa cagatgtgca tgatgatcac tggccaattg cgtcacttct ctcaaagtca 16260 gtctgtgatt ggtccctgag catctgtcgg tcagtttcat gcacagactg caaagcatat 16320 ggttttgtct actctttgtc tctcagtgat aaacccacat ggcattttgt aaaagtggat 16380 acatcaggcc aggtgtggtg gctcatgcct gtaatcccag cactttggga ggctgaggca 16440 ggtggatcat ttggggtcag gagtttgaga ccagcctggc caacatggtg aaaccccatc 16500 tctactaaaa atacaaaaat tagctggatg tggtggcagg cgcctgtaat cccagttact 16560 ggggaggctg aggcaggaga attgcttgaa cccaggaggc agagcttgca gtgagccgag 16620 atcatgccac tgcactctag cctgggtgac agagcaagac taccatctca aaaaaaaaaa 16680 aacaaaaaac agtaatcaag catgaaaatt atgaaatgct cagagataaa atgcgcgagg 16740 cctgtacact gtaatctaca aaacactgct gagagaaatt ttaaaagacc taaataaatg 16800 gcaagttata acatgctctt gaatcagaag actcagtatc ttaggatggc gacttttccc 16860 aaaatgatct acagattcaa agcaatcgga atcagacctc agcatgccta cttgtagaat 16920 ttgataacct gattctaaag tttatatgga aatgcaagga acccagagtt gctaaaataa 16980 ctttgaaaaa gaacaacaca gttgaaggac ttagactaca tgatttcaag aattattata 17040 aagctacagt aatcaagaca gtatggtatt gatatgaaaa tagaccatta gatgaatgga 17100 acagaatagc aagtccagaa atagatccac acatatatgg tcaattgatt ttcagcaaag 17160 tgccaagtca tttaagtggg gaaaagataa tcttttcaac aaatgatacc ggaacaactg 17220 gatagccata tgcaaaagaa cctcaacctt cagctcacag cactacaaac tcataattat 17280 tatcattata ttatactatt atgtaataat agtatatatc atgttacata ttatattatg 17340 taatatatat tatatgatac tgttatgtca tataattatt attgaaatgg gtcatagatc 17400 taattgtaag agttaaaacc atccaggtac agtggctcat gcctgtcatc ttgcactttg 17460 agaggccaag gcgggtggat cacttggccc caggagttac aagaccatcc tgggcaacat 17520 agcgaaacac cgtctctaca aaaaaatgaa aaaattagtt gagcatgatg acactcacct 17580 gtagtcccag ctgcacagta gtctgaggtg ggaggatcac ctgagcccag agaggtcaag 17640 gttgcagtga gccatgattg caccactgca ctccagactg ggtgacagag agaccgtgtg 17700 ttaaaaaaag agttaaaact ataaaacctt cagaagaaaa catatgagaa aattctagtg 17760 atttggggtt tggcaaagat tccttgaaca tgatttaaaa agcattaact aggccaggta 17820 tgctggctta cacctgtcat tccaatgctt tgggggaccg aggtgagagg atagcttgag 17880 gccaggagtc cgagagcagc ctgggcaaca taacaagagt gggtctttac caaaaaaaaa 17940 aaataaaaag cctgtgccag gcacagtggc acatgtctgt agtcctagct actcacgaag 18000 ctgaggcagg aggatcactt gagcccagga gttgaagctt gcagtgaatt atgaccatgc 18060 cactgcactc cagcctgggc cacagagtaa gactaagact cagtctctta aagaagaaag 18120 cgaccgggcg cagtggctca cgcctgtaat cccagcactt tgggaggctg aagcaggtgg 18180 atcacaaggg caggagatga agaccatcct ggctaacacg gtgaaacccc atctctacta 18240 aaaatacaaa aaattagccg gacgtggtgg taggcgcctg tagtcctagc tactcgggag 18300 gctgaggcag gagaatggcg tgaacctggg aggcggagct tgcagtgagc caagatcgta 18360 ccactgcact ccagcctgga caacagagcg agactccatc tcaaaaaaaa aaaaaaaaaa 18420 aaaaaaaaaa gaagaaagca taaactataa aagaaaaaat taataaatta gtcatcctca 18480 aaattagaaa cttttactca tcagaaaaca cttaataaaa tgaaaagtca agccatagac 18540 ttagagaaaa tatttacaaa acatatatct gacaaaggac ttggatatgg attatataaa 18600 gaactattgt aattcaataa gatgtcaaac aacccaatta aaaatgggtg aaagatgaac 18660 taactcttca acaatgggca tgtcatttga atggatggta agcaagcaca tgaaaagatg 18720 ttcatgtgcc tttccctcat tagtcactag ggaaatgcaa gttcatagac atctctcttg 18780 gtagaaagat atcactacac acccacaaga gtggctgtaa ttaagcagtc tgaccaagta 18840 tgcgtaagaa tgtggaataa gaactctcat acactgctga tgggaatgta aaatgatagc 18900 cactttggaa aacattttgg caaataatac cacttacatt attatcgaaa atattgtata 18960 cctgaaagaa ctcaaagtga aaaagctata tactgtctgc ttccaaggct acacattatg 19020 ggaaaggcaa aactatgaag acagtaaaaa gatgcgccag tggttgccag gggctcatgg 19080 ggagggaaag aggaatgaat aggtggaaca cagggcatgt ttagggcagt gaaactattc 19140 tgtatggtac cgtaacgatg aatacatgtt attaggcatt tgtcaatacc cataaaatgt 19200 acaacacaaa gagtgaaaat gaaaactgtg ggcttcagtt agcaataata tgtcaacatt 19260 ggctcatcag tggcaacaaa tgtacctcac caatgcaaga tgtttgtttg ttgtttgttt 19320 gttttgtgac ggagggggtg cagtggcgca atctcggctc actgcaagct ccgcctcccg 19380 ggttcacgcc aatctcctgc ttcagcctcc ggagtagctg ggactacagg cgcccgccac 19440 cacgcccggc taattttttg tatttttagt agagacgggg tttcaccatg ctagccagga 19500 tggtcttgat ctcctgctgt cgtgatctgc ccgcctcggc ctcccaaagt gctgggatta 19560 caggcatgag ccatcacgcc cggccaccaa tgcaagatgt taataacagg gaaactgtgg 19620 tgggagtgag gtggtatatg agacctctct gtactttcca ctcaattttt ctgtaagccc 19680 aaaacttctc taaataagaa agtttattaa ttaaaagtta cttttatagt gtatctatat 19740 ctaggaataa atctgaaaaa gatatataag atctctactc agaaaactga ttatgttatt 19800 aagagagctt aaatatagcc caaataaata gatggatata ctatgttcat ggaagggaca 19860 gctcagtatt aggaaggtgt cagtcatctt aagaaaagcc tcatgtgtca cacaagggat 19920 actgacatct gacaccaagc acatgtaggc atcctgacta cgtttacttg aatgatgtgg 19980 actttacaga gctgactata gacagttcaa atggcctgaa aactgttcaa tgcactccct 20040 cccaggctgt catgggatgc acttcaggaa ctttactttt taacaagaaa attcagtttt 20100 cctcttaaac agctggcttc tgttccatta gcattcttgt cactttaagt tgcattcatc 20160 tttgtttttt ttttttagaa aaacatttgt tctgcaacca gtcttgtcct ttaaatactt 20220 gtactgtata caggctcttt ttcataggtc cattacttaa aatgatgtaa gtgtgttttt 20280 ggtggcaggg gggtgggagt tgtttgtttt gttttgttga gacacggtct tactctgtca 20340 cccaggctgg agtgcagtgg tgtgatcttg gctcactcct ggcctcaagt gatccaccca 20400 cctcagcctc ctaagtagct gggaccacag gtgtgtacca ccacacccag ctaatttttt 20460 tttttttttt tttttttttt gtagggacgg ggttttgtca tatcacccag gctggtctca 20520 aactcctgga ctcaagggat cagcctgtct cagcctccca aagtgctggg attacaggtg 20580 tgagccactg caccggtcct gatttgagtt tttgtaagac agggaacaat gttcagaatt 20640 tagcaccaat gtcagactca ttctgtaaat ttttattgaa cgtctgcctg gtgtaggaga 20700 ggaagatgac agacaagaat tcttcctcca agagttacag gtcagttgag cagaaaaggc 20760 atacatcaat acccacaatg agagttgtcg tgattcagag gagggacaaa gtccttcccc 20820 tggagggatc ctgagcactt tggagaggaa aggcatctgt actgcccccc aaatgtgtag 20880 aatgggatgc attcctggca gaaagaagta ggataaagta cagaggccag ggctgggtgc 20940 agtggttcac gcctgtaatc ccagcacttt gggaggccga gacagcagat cacctgaggt 21000 caggagttcg agaccagcct ggtcaacatg gcaaaaccct ctctctacta aaaatacaaa 21060 aattagccag gcacaatggc aggtacctgt aatcccagct acttgggagg ctgaggcagg 21120 agaattgctt gagcccagga ggcagagatc gcagtgagcc aagactgcgc cactgcactc 21180 cagcctgggc aacagagcaa gactctgtct cataaaaaaa gaaaaaaaaa aagtacagag 21240 tccaggaagc ctggggtggg gctggcagat gccgagtcat ctattttggc cagagttcaa 21300 ggcttgctag gggacatgaa gagaagattc gtgcattcta gttcaaactc caccagatat 21360 ttgagctcct tctctgtacc aggcattgtt ctaagatacg taagtgaaca aaacccatga 21420 caccctcgtc tatgagagct gatcctctgg cagggacaga caggtcatga gtggagtgat 21480 ggagcagctg gcctggtgac ttagccgcct tcaggtacag taggaggagc aagcccagga 21540 caggtgagtg ggtcaagggt gccagaaggg gtgagggcac caggaagctg gtccagtttg 21600 gcttccctga ggtggtgacc aggacctagc atctgaggaa gggctggaag caggtgagag 21660 caggtggagc agacatcagg atgggagcat cctgacaggg agggcagcag ggtgggctca 21720 tgagaggaac agccaggaag tgtgactcga gcagtgtcct ggagaggagg aggaggagaa 21780 agaggtcagg aggtcccagg ggagaggcag gaccagtctc gtggaggtcg gggccgttgt 21840 gaggactctg gtttgtgttg tgtgtgaaag gccatgggat ggggaccagc gagggcttct 21900 taggggactg gatatgctct gatctagctg ctaaaaagcc cccttgggca gcttgcaggg 21960 cccgggcaga agctataggt ggttctgagg tttgcagagg ggcctgaagg ggtggggccc 22020 ggccaagcaa ggtggctaag tgggaaaggc tccaccgcgt tgggtgtagg aagaccttga 22080 ccttagctcc agcccagcca ctgagcagcc gtgtcgcctt gggtgatacc tgtccctggt 22140 cggtttccct acctgtgaat ctgggtactt ggaagccatg ctcgagaaga gcccatcccc 22200 aggaggtgat cagggttctc ctccaggtga ggaacctggc agccgtgtgt gagaacctta 22260 gaaaagggag agggaagagg ctgtggcagg aagtgaggag ggagttagtg ataccctggg 22320 caggatgcca tgagctggga tggaaaccac aggatgaatg caagtaatta aaaaaaaaaa 22380 aaaaaaaaac agcattgggc cgggcagtgg ctcacgcctg taatcccagc attttgggag 22440 gccgaggtag gtggatcacc tgaagtcagc agttggagac cagcctagcc aacatggtga 22500 aactgaaaat gcaaaaatta gccaggcatg gtggcgtgtg actatagttc cagctactca 22560 ggaggctgag acaggagaat cacttgaacc tgggaggtgg atgttgctgt gagctgagat 22620 cgtgccactg cattgcagcc tcggtgagag agcaaggccc catctcaaaa aagaaaaaaa 22680 cagactttcc gaccaaacga tcgacaaacc agactgtcca aacagccata agccgtaact 22740 ttgtgcggag gtaaaagacc gaggtcacat cgggacctgt tggattcaag gcatgttgac 22800 agctgtttcc aggcttcaga tagagcctcc agctggcagg gtggccacag ggcttgttga 22860 gtaggaagcc tcgttgcttt gacaggttac ttggccccat gagggacaat cccatagtca 22920 gttacccaga aacgtgactg tctccttgaa atcctcagca tggggtctta tgaataaacc 22980 cttactagat ttcctgttct gtcttatttt tatgcagagc tttactttat agcagaaaat 23040 tccattttta cccttaaatg gcttgcttct gctcccttag tgttcttgtc actttaagtt 23100 gcattcatct ttgtcccttt agaaaaggat ttgtcctgca accagctctt gcagaaggta 23160 cttggtttat tgttaaccga tgtttgctaa atgtttgaat tatgttgagt tgcttaaagt 23220 catgctatcg ggtagatgtt gtggctgttc ttttcactct cttatttggg gatttacaaa 23280 acagttatgt ttttagtttt cttttatttg ttgtgttgaa taggaatgta gctctgggaa 23340 cctctagttc caaataagaa agccttggac acatttccag ttggcaagct ggcaaaatga 23400 agggcgtaca agttgttaga gaggctggga gcctatttaa gcacccagct tcaggatggg 23460 acatgggata tacctcgagt tagaggttct tattaactgt ggattcttct atgcagatat 23520 ctgtcacaat ataagttact ataagtcagt actaaggcag ctgctacatt ctgtttgcca 23580 aggggaagaa gaaagcttgg aaatggtatt ccttaaaaat gtcagtatca taaaagacaa 23640 agaaaagctg cggaaatgtt tcagattaaa agagagaaga caataaaatg taatacctga 23700 ctctgaacag catccagtac tgaaggagga aaaatgctat caaggacatt attgggtcaa 23760 ttaacaaaat ttgaatacga atcatagatt gaactgtatc tgttaaatta acagaagcga 23820 agtgttctgt ggtgtgtagg agcacactgc cattcttagc aaacgtgtag tttagtattt 23880 aggagaaagg gccatgaggc atgcaactca ccctcaaata cacacacaca tacacatata 23940 tacatacata cctataaaga aagaaattat gggctaggtg cagtggctca tgcctgtaat 24000 cccagcactt tgggaggccg aggtgggtgg attgtgaggt caggagatcg agaccctctc 24060 tactaaaata caaagaatta gctgggcgtg gtggtgcacg tctgtagtcc cagctactcg 24120 ggaggctgag gcaggagaat tgcttgaacc caggaggcag aggttacagt gagccgagat 24180 tgcaccactg cactgcagcc tggcaacaga gcaagactct gtcttgaaag aaggaagaaa 24240 gagagagaga gagagagaga gagagaggga gaaagaaaga gagagagaaa gaaagaaaga 24300 aggaaggaag gaaggaagga aggaaattat gataaagcag atggttaagt tggtaactac 24360 cagtgaatat gggtaaagtt aggatgttct ttactctgtt ttgggggtgc aacttttcta 24420 taagtgaaag tacttccaaa taaaaagtta aaaggcaagc aaataaataa aagagacagt 24480 ttctatgtta tatatcctag ctatgtttac catgtctgga ttctgaaagc tgcagagcag 24540 aaaacctgaa gaacagatca cctgttctta aaataccact gttggccaga catagtaact 24600 cactcctgta atcccagcac tttgggaagc cgaggtggga ggatcacctg agctcaggag 24660 tttgagaaca gcctgggaaa catagtgaga ccctgtctct acaaaaattt aaaaaattat 24720 ccaggcatca tggttcgtgc ctgtagtcct agctactcag gaggctgagg taggaggatt 24780 gcttgagcct gggagttcga ggctgcagtg aaccatgatc acactaaagc actctagcct 24840 gggcaacaga gcaagaccct gtatcaaaaa aacaatcaaa caaaaaatca ctcctaattt 24900 tcctcccttt tagtactttt aaaaattaac ttaaaacatt ttttggataa ttgtagtttt 24960 ttttactttt ttttttttga gacagagtct cattctgtca cccaggctgg agtgtactgg 25020 tgcaatctca ggtgactgca acctctgtct cctggattca agtgattctc ctgcctcagc 25080 ctcctgagta gctgggttca taggcgtgca ccacacctgg ctcgttttta tatttttagt 25140 agagatgggg tttcaccttg ttggccaggc tggtctcaga ttcctgactt caagtgatct 25200 gcccgccttg gcctcacgtg cagttttagg aaataataca gagatcccca gcactctttc 25260 cagttttccc caagggtaac atcttgcaaa gtgagaggac gatatcacag tcaggatact 25320 gacattgata ccatcaagat acataatgtt tccatcacca atcagtggtc atggtgcctt 25380 ttatagccaa acccacttct ctcctacctt cccatccctt ttttaatttt gccagtcatt 25440 aatctgttgc ccatttctgt cattttatga atgtcacata ggccgggcgc ggtggctcac 25500 gcctgtaatc ccagcacttt gggaggccga ggcaggcgga tcacgaggtc aggagatcga 25560 gaccatcctg gctaacatgg tgaaacccca tctctactaa aaaatacaaa aaattagcca 25620 agcgtggtgg cgggcgcctg tagtcccagc tactcgggag gctgaggcag gagaatggtg 25680 tggacccggg agacggagct tgtagtgagc tgaaatcaca ccactgcact ccagactggg 25740 tgacaaagcg agactccatc ttaaaaaaaa aaaaaaaaga atgtcacata atgaatcata 25800 tggcatataa ccgtttgaga ctcagggtaa ttctcatgag actcatccag cttgttggtg 25860 catcaacagt ttattccttt ttattgctga gtaatttcca tggtatggag gaaccatggt 25920 ttaactattc acccattgga ggacatctag gttgtttcca gcttggagtt attatgaata 25980 aagctgctgt gaacatttgt gtacaggttt cttggttttc tggtttgttt taaacagttc 26040 tagccaggca cggtggctca cacctgtaat cctaacactt ggaaggctga ggtaggagga 26100 ctgcttgatc ctaggaggca gaggttgcaa ggagccgaaa ttgtgccact gtactccagc 26160 ctgggcaaca tagcaagacc ctgtcattca taggtaggtg gatggatgga tggacggacg 26220 gacagataga taggtagaaa tgtaaattac agggctacgc tcagtggctc atgcctgtaa 26280 tctcagcact ttgggaggcg aaggcgggcg gatcaccaga ggtcagcagt ttgagaccag 26340 cctggccaac atggcaaaac cccatctcta ctaaaaatac aaaaattagc caagcatgct 26400 ggcatgtgcc tgcaatccca gctactttgg aggctgaggc aggagaatca cttgaaccca 26460 ggaggcggag gttacaatga gccaagatca tgccactgca ctccagcctg ggccacagag 26520 tgagactccg tatcagtact ttctttttat tgtttttctg ttattatagt ttaagttcat 26580 tgttattaga ttatatactc tgtatggctt caattctttt aaatttgttg aggtttgttt 26640 aatggtcaaa gacatggtct gtctaggtga atgttccatg ggcttttagg gaaaaaagta 26700 tattctagtg ttgttgaatg gtgtcttagt ccattcaagc tgctataaca aaataccgta 26760 aactgggtga tttataaaca acagaaattt ttctctcaca gttctggagg ctgggaagtt 26820 caagatcaaa gtgccagcag attcagtgtc atgtgaggac gtgcttcctg cttcatagat 26880 aagaggtaca tacacgttta ggagcatcgt gtcttcctgg tggatgaatt ctgttatcat 26940 taggtgatcc tttgagcact tttaaaaaga atctgttggc cgggcgcagt ggctcacgcc 27000 tgtaatccca ggactttggg gggccaaggc gggcagatca cgaggttagg agattgagac 27060 catcctggct aacacagtga aaccctgtct ctactacaaa tacaaaaaaa ttagccgggc 27120 atggtggcag gcgcctgtag tcccagctac tcaggaggct gaggcaggag aatggcgtga 27180 acacaggagg cagagcttgc agtgagccaa gatcacgcca ctgcactcca gcctgggcaa 27240 caaagtgaga ccctgtctca aaaaataaaa taaaataaaa taaaaataat ctgtttaata 27300 gcctactagt gttcttcctt tactatttta ttgagcatta attaatccca acattatgtc 27360 tatgtcagga ctgatgacaa tatttggtat aaaaatttga tagtctcaga ggctgaggca 27420 ggagaatgct tgaatccagg aggcagaggt tgcagtgagc tgagaccgtg ccactgcact 27480 ccagcctggg caacagaaca agactccatc tcaaaaaaaa aaaaaaaaaa atcgatagta 27540 tcatatcctc caggattcaa agtgaacttc aaacagtctt atgtagtcta aattttggaa 27600 tgcatcccag tattgagttg cagcagggat ttgagttttt gtgaagagag agaggtatat 27660 cagaatcttg ggcataaact aaggagccat gtcagaacct caggtgtatg ccaatgagat 27720 agatcagaac ctcaggcatg tacccgatga gacagatcag aacctcaggc gtgtacccgg 27780 tgagacaggt cagaacctca agcgtgtacc tgttgagaca ggtcagaacc tcaggcgtgt 27840 agccagtgag acaggtcaga acctcaggtg tgtacccagt gagacagatg agaacctcag 27900 gtgtgtaacc agtgagatat atcagaatct tgggtattta cccaaagagg tatagcagag 27960 tctcaggtat atactcaaga aggcatatct tgaggcttta agtatctagc taaggattta 28020 tatcaggatc tcaggtttat acccagggag gtatagcaga atttggggta tagatctaag 28080 gaggtctatc agtctagagc atatagccaa ggaactatat cagaacctca ggcacctacc 28140 caaagaggca ttttaggact cgtaaggagg gggtagattt caaaagtgta gtctaacagt 28200 ttatctactt tgaaatttaa aacaatatta aaggaaaaca tgaaatattt ctatctgtca 28260 gaaggtgaca tgagttttaa acaattaaga aatatactgg ctgtggcctt gtaaccaaat 28320 tattatgcct atagaaatta cagactccat tttccaggat agaataacag ggactgactt 28380 accttctcat ctgagataac aaaacctcca tacaaataca tgaaacaatg ttcttcaaga 28440 tgctggacat caggcagtga agggcactga tggttgtaag acaaggtgag aggtgtggct 28500 tgagagagtt tccaggttgc agtgcaggga gaggggaaac tgaggcagat cttggcagac 28560 ttcctcagtt gacaaaatag agctgagagt ccagggagac catggtgtat agattatcca 28620 aagcaaagta tgagaggtgc aagccatata cagagggact ccagagatct accaaagtac 28680 ttcttggtgc atccatatga gcaaaactac ttgaggccag gaaaagaacc atctgagagg 28740 attagaagga acagtgccca gtacttgtgc cagccaggaa tggtgcctga tactcacgca 28800 gggccaggaa cagtgcaggg atgtgagtgt ttgttaggag agggaggtat atcagaatct 28860 tgggcataaa gacaagaaac catatcagaa catcaggtgt gtaccaatga gatagatcag 28920 aatctcgggt gtatacacag tgagatagat cagaatctca gatgtgtaca cagtgaagca 28980 gatcagaatc tcagatgtat acacagtgag atagattgga atctcaggta tgtacccagt 29040 gagtccaaga gcatggtgct ggcatccggt gagggccttc ctgctggatc gtgacatgaa 29100 gcaaggcaaa gagcctgtca gctcagggct ctcttcctct tcttataaag tcaccagtcc 29160 tatcatgggg gccccaccct gatgatctta taatcctaat tacctcccaa aggctacctt 29220 caaatgctat caacatatga atttggaaac taagtttcca gcacatgaaa tttgggggat 29280 acattcaaag tatagcaaat attacatcat aaccagtagg attcatccca ggaaatgcca 29340 aatggcttga taatcaaaaa ttaatgtaac tcatcgtatt aacaggatga aaaagaaaaa 29400 ccatgtgatc atcttagtag atgcagaaaa gcagttgatt aaatcccaca ttcatttcta 29460 acttaaaaaa acaactggat tttgacagag gtgcaaaggc aatttggtag agaaaggaca 29520 gtcttttcaa taaatggtgc tggtgcaatg gttatccata tgcccaaaat gaactttgac 29580 ccatgcctca tgccatacac aaaaattaac tcaaaataga tcagagatct gaaggtaaaa 29640 tttaaaacta taaaacttct agaagaaaac acaggagaaa aatctttgtg accttggtct 29700 aggcaaagat ttcatagata tgacaccgag aacacaatct atgaaagaaa aaaatcaata 29760 aattgaactt catcaaaatg aaacttttac tgttcaaaag acagttttag gagaatgaaa 29820 acacaagtta cacattggga agaaatattc gaaaagcatt tgcctgataa aggtattgta 29880 gctggaagac agaaaaaatt ctcaaaactc acctagaaga aaataaccca gttttaaaaa 29940 tgggcaagag atctgaacaa acacattgtc aaagaagata gatgaatagc aagtaagcat 30000 gtgaaaaatt ctcaatgtta tcagtcatca gagcaatgca gatgaaacct acagtcccca 30060 tgctaatgtt ctacaactta cacagtggtg gtatgatacc actacatgcc catttgaatg 30120 gccaaaatta gaaaggttga ccataccaaa cattagccat gatgtgcagg aactagaact 30180 ctcatctttg ctgacaggaa ggtaaaatga tacaaacaca ttgaaaaaca ggttggcagt 30240 tgcttttttt tttttttgag atggagtctt gctctcccag gctggagtac agtggcgcga 30300 tctcagctca ctgcaacctc tgcctcccaa tgaattagaa aaataataat aaaggtaaca 30360 atagcagtaa taataataga aataatgata gtttctttaa taaaaatgct gtttaggccc 30420 agactgaaag gctttaagta accactcccc cactgaagtt agagttaaga aagaatatta 30480 attttccttg tgtgaaacat taatcttatc tagcctccat gtattttgta agttctgtaa 30540 attcctgttt tccctgcaca gctgcaagtt cacaaggcag ataagcttaa gctgcaaaac 30600 atgtttttct taagatgtaa ggcatgtcac aagaatatca caagatgata acggccttta 30660 ttctcacttc tgtatgcctg cttcctgcct cacatatttc ctgcctcaag atgcgtaaaa 30720 ggtacttgcc ttctttgttt ggtgctctga ctttctggat gcaagtccac tgagccagtg 30780 tacaccttaa ataaatcctc ctgaacccca tcaatcgctc cagttctctg atttcccact 30840 acattttctg ggggctcgtc cgggattgga gatggcagat tttctgtctc ccttgcctgt 30900 ggaactggag cccgggtcga gggagacctg ggacctttgg tgccaatggg aggactttag 30960 cccggaaagg agattggctc tcctgcatcc cggtgtcctt cctagacagc acaacggaac 31020 ctataaaggg gttgcaggac ggttccagca ggggctgggg atggtgagag tagctcactg 31080 attcagatga cagggttttg ccatgttgcc cagacccaga ggggctgggg acagtgagag 31140 tagctcactg attcagatga aacttacacc ttagccgatg caggacacga gagtggctca 31200 ctaagttggt caggaaagaa actgaaaatg ggaagagtgg cttcctgcct tgactaagga 31260 tcgggaactg ggagcgggga ggtgtgtgaa agagatggtt ccgggagggc cgtgatgtgg 31320 ggagacacag atctcttagc acggactgtg tgctctgagg cgagtgtgtg attgaccaga 31380 accagggcat cacatacagc tgacaggagc tgccccacag ctgcagcagg ctgtggcagg 31440 aataaggtac tctcctagct aagcagcacc tgaaacttcc gtaataggac ccagtctggt 31500 cagtctggaa cgaaagtgag agtgagtgtg catcacaaag ggcgggatgg gaggaaaagc 31560 atcgaaaccc actcctctgg ggtgcatgtt aaagaatttt aagaaaggtt ttgctggaga 31620 ttatggaatt aagttgtccc ccccaaagat tgagggttct gtgtgaagtg gaatggcctt 31680 cttttaatgt cgggtggcca gccgagggta caataaatag ggaaatgatt ggtcatatat 31740 ttagggtagt gactggggtt ggaggacacc ctgggcatcc agatcagttc ccatacatca 31800 attcctggat gatcacagtc tagacatgcc ccaaatggtt acagccttgt ctggcaactt 31860 actgtaagac tctagtgacc tgagccgaac ctaaggcagt tagagggccc ccttcaccag 31920 acacctcagg tggaaagaaa aagccacagg aaaattagga aagacctgtt ctacttcact 31980 gggatcaagt gattctcctg cctcagcctc ctgagtagct gggagtacgg gtgtgcacca 32040 ccacgcctgg ctaatttttt taaattttat ttttagtaga gacggggttt tgccacattg 32100 gccaggctgg tcttgaactc ctgacctcag acagtctgcc tgccttggcc tcccaaagtg 32160 ctgggattac aggtgtgaac caccatgccc agccagcagt ttcttataaa gttaaaccaa 32220 tgcctaccat gagatctggc aatcccactc ctaagtattt ggccaagaaa aaagaaagca 32280 tatattccat acagagtcta gtcctgaatg tctatagctg ctttatttat aatagctcag 32340 acttggaaac cattcagatg ccattaatag gtgaatatat tctcaaactg tggttatcca 32400 tacaatggag tattactttg caatcaaaag gaatggccta tgaataccca taacaacatg 32460 gatgaatgct gaaataattg tgctgagtaa aagaagacag gaaaaataag tataatacat 32520 actgcttgat tctatttgta taaaactaga aagtacaaac taatctgtaa tgacaggaag 32580 cagaccagtg acagtgggca tggaggggca agagggagag attagatggg cacaggagag 32640 ctttgaggat gatgggtctg cgtactgtct cggctatgat agtggtttca caggttgata 32700 catacggcaa aaaataccaa atttgtacac tttaaatatg tacagattat tgtatgccag 32760 ttacatgtcc ataaagcttt cttttgttgt tttgttttta ttttattttt tgagacagag 32820 tctcgctcca tcgtccaggc tggagtgcaa tggcaccgtc tcagagcact gtaacctccg 32880 cctcccgggt tcaagcgatt ctcatgcctc agcctcccaa gtagctgaga ctacaggcat 32940 acgccaccat gcccagctaa tttttctatt tttagtaaag acagggtttc gccatgattg 33000 ccaggctggt cttgaactcc tgacctcagg tgatccaccc acctcggcct cccaaagtgt 33060 tgggattata ggcatgagcc acagcaccgg gcccataaag ctgtctttta aatgaaaaaa 33120 agttgtcttg aaataagcat tagaactgtg gctttggctc tgaaatcctc atctgaggac 33180 ccacactcgg gtgccccaat gtggcggtgc ttacagaaat gactccatct gctaaatgag 33240 taaatgggta attctccact gaacacacac tcgtttagca gcataagcag caagagttca 33300 ggtaatcctc acattgcaat ttgtcattag tttaaacttc cagtctttgt tttaaaaaca 33360 cattagaata atactacatt ttccctcatc tctaaacttg actgaagact ccaagagaga 33420 gtaatattca tcaagaggat catctactca acacagataa actgggaaag aaaaataact 33480 tgtgagtaat tcagaatctg gattatcagg tcaggctcaa tggctcacgc cagtagtccc 33540 agcactttgc ggggcccagg agggcagatc acttgagttc aggagtttga gaccagcctg 33600 ggcaacatgg cgaaaccctg tctatacaaa aaatagaaaa attagccagg catggtggca 33660 tgtgcctgta gtcccagtta cccgggaggc tgaggtggga ggatcacttg agcctgggag 33720 gtcgatattg cagtgagctg taattgcacc atgcactcca gcctgggtga aagaaggaaa 33780 ctctgtgtcc aaaacaaaac aaaacaaaac aaaaaaagct aaattatcaa atgtctagat 33840 cgttgatggt tggaagtaaa gttgagaaat gttcacactg ggagatgaca cacagtaaac 33900 cacacagagg gttctaacgt ggttgttaga agcagaaact agaggcttgc tgcctgaggt 33960 caaaccccgg tcccggtggt tctgtgcccc agcgcatgtt gtggtagcct ctctgtactt 34020 cagtttcctc atctgtaaag taaacataat gataatgcct gcctcatggg gttgctgtta 34080 ccaggaagtg agttaatgca cattaggttc ttatttatga cagtgcctgg cataggataa 34140 gggctcaaaa agtgttagct ggaactacta tcattatcaa catctctaat ttattgcagg 34200 gttggatctg aaaaatggct gatgatgatt tgatgatgac ttcattttta taaaacaata 34260 atattgcagt gcaaattaaa cacaagcaac ctgcaacacg ccactgcaag ttggatgtct 34320 agaaaaggtg ccatgagtta ccttctaaaa catcataaga aaccatgttc accaataatt 34380 accataatag gagagaagta ccaagtacca tggggagaca gagtccagaa tctcagagag 34440 agacacagtt accttttagt tacactaatg gggaaaacag gagctttgct acccttgcac 34500 ctgatggagg gcttctctga atatagggct atggggtcag aagccagttt cctcccatat 34560 ttagaaggtt tccaactgtt atctttcact tcccatgttg ctgttggaaa atccaaaagt 34620 attctatttg gccaggcaca gtggctcaca cctgtaatcc cagcaatttg ggaggccgag 34680 attacctgag gtcggaagtt caagaccagc ctggccaata tggcaaaacc tcttctctac 34740 aaaaaataca aaaattagcc aggcgtggtg gcgcacgcct gtagtaccag ctattcggga 34800 ggctgaggca cgagaattgc tttaacctgg gaggtggagg ttgcagtgag ctgagattgt 34860 gtcactgcac tccagcctac gcgacagagc aagactccgt ctcaaaaaac aaaaaaagta 34920 ttctgttgga tcgtttgtgt gcgacgtgtt tttccctctc agaaagctcg tagggtcttc 34980 tctttgtctc cagcatggtc tggaatttcc cagtgagtga ccatctgtgt gtgtgtattc 35040 atccattcca tggagtccct gctgggccct tgcaatctgg aaattcatgc ccatcatttc 35100 tgagaagtta tcctgaacgt actggttggt ggtttcctgt gctccatgtt cttgcttcct 35160 gctcttcgga gctcctgtta tttggttgag ttttgtctcc tggactggtc ctctcttact 35220 tctcttgctt ctcccatgtt ccatctgttt tcactctact ttctgtgaga tcaggccctt 35280 tatcttccaa cccttctatt aggtttgcaa ttgagtttgt aattccctag aaaagttctt 35340 attctctgaa tatccctttt gatagcatac tcttcctttt tcatgagtgc agtgtttctg 35400 catggctctc atcagaacat gctagtgtct cccatttctc ataactttct agagtgagga 35460 ccatttgaac ttggagtgcc ctcattttaa aactctgtgg ttgaccttgt tcccctcttt 35520 tgctgctgct tttcaccaga ctttgaagaa gcagaagtac attcagaact tgtctgctct 35580 ggcaaaagac aatactgttg gcttaatcta aaaattgaag aagaaagctc aaggagagaa 35640 gtttaaaaat ataccacctc tggctgggcg cggtggctca catctgtaat tccagcactt 35700 tgggaggctg aggcaggtgg atcacctgag gtcaggagtt caagaccagc ctggccaaca 35760 tggtgaaacc ccgtctctac taaaaataca aaaattagcc aggtatggtg gcagccgcct 35820 gtaattccag ctactcggga ggctgaggca ggagaatcac ttgaacccag gaggcagagg 35880 ttgtggtgag ccaagatcac gccactacac tacagcctgg gtgacaaagt gagactccgt 35940 ctccaaaaaa aaaaaaaaaa aaaaaatata tatatatata tatatatata tatatatata 36000 tatatatata tatacacaca cactaacctt cagcatatag gactattgca gaagggatta 36060 tctttctact tggttggttc ctcttggtct tgagcaaatt ttgacttccc tgagttggcc 36120 cctaaaagtt aaagggaaag ggccttttct ctttccttta aaaccaatat ggcatatttg 36180 ctgagaactt agataccaca ggattggcag tgtagactta cattcataga ccggatgcca 36240 tcagccaacc ttgagtaatt tgcagcacac tgcatcattt tatttaagta atgcgaagtc 36300 cttgacatgt ctcagacatt gtcttggtta cttgtaaggt ctcacataaa tctaattttc 36360 ctctttctct gccctttctg gttcagctca gtttattcaa gggtgtattt gtgcaacaca 36420 cttgaataag gtgtggtccc gcctttgtag atgttatagt ttgggaagac cagccgggca 36480 gagagaagag catgattcaa ggatgaaggc gtgggctggg ggccgaggga gcaaggattc 36540 ccagtaacga gggaggaagg agcagcacca tgtgcccatt actctataga catctcgaac 36600 cacctggcca tgtagctgtc attaacctaa atttacagtt attgaaactg aggttcagcc 36660 aggcgtagtg gctcacgcct gaacacagga ggcggaggtt gcagtgagcc gagatcacgc 36720 cactgcactc caggctaggt gagagagcga gactctgtct caaaaaaaat aaataaataa 36780 aagaaaagaa caaaactgag gttcaaagaa atgtacagtg ctccccccct tatccaaaga 36840 ggatacactc caagccccca cagtggatac ctgaagcctc agatagtatc aagccctata 36900 tatgctatgt tttttccctg tatatgcata cctatgataa agtttataaa ttaggcacag 36960 taggagacta acaacaatga taataaaatg gaacaattat aacatacact gtaacaaaag 37020 ggtctcttcc tctctctctc agaatatctt attgtactgt actgggggta actaaaacca 37080 aggaaagtga aaccatggat aagggatatc tactgtataa ggtggagttt tcaaggtcat 37140 agcactgcct tcccctgagg ttggccttgc agcctctcta ggcactgctg ctgctgctaa 37200 gaacccctgt gaggtgaaca ctgtaagcat catcattgct tctcagaaga ggagacctgg 37260 cttacagagg tcaagcctca ggtaccttaa acaccatttt aaaactgaac tcatggccag 37320 gtgcattggc tcatgcctgt aatcccttct ctccatgctc aaaacctgcc ctccttgtct 37380 ttatattcca aatttcgtgg gtgccacctc ctctgcccag tgacttaagc cagagcatac 37440 attcatccta gactctgtcc caggtccctg gtccaggcag ctgccagttg tcaggatcag 37500 ctctttatct cgcagtcctc ctgcctcttg tgtcattacc cagggctgtc accatctttt 37560 cttgggacag ttacaacagc cccgtaagga gttgtgctgc ttctagtctt gttccctttg 37620 aatctggatt ccttcttgcc atcaagacaa tcctgataca aatctgatca cgtcacactt 37680 cccttcaata gtcttccatg gctccttatt gttttaggat gaaatccaaa ctcctaaaca 37740 tggggattaa caatgtgcca tgattggcac tgctggcctc tcctacctct gcagactcac 37800 ctcttgccac ttctcccttg aggtagatca aaaatggtca caagttcttt gaggctcttc 37860 ccatcaagag gtagagttta tttccccacc tcttggatct ggcttgcctt gtgacttgct 37920 ttgacccaca gaacgtaaca gaaaggacac tgcctaactt acaaatgagg tctaccttaa 37980 gaggctttgc agattccaca ttcaacctct tggaatgctg ccaccatctg agaagcctga 38040 ggtggcctct gtgaggatga aagacttcat ggtgagaaat acctagcgaa cagcctggca 38100 ccagctacca ggcatgtgac tgaggccatc cagccatagc tgagccacaa aatgaccaca 38160 gctatgtgaa ttatcccagg tcagaccagt agaagagcca cctggctgag ctcagcccaa 38220 tttgctgacc catagaattg tgaacaaata aaatggttgt agttataagc cattaagttt 38280 cagagtttgt tacacggtaa catgtaactg atacaactct tggagccagt tgttcagcca 38340 ttctcaacca cttattcaat gatgttttgg gccatatatg cagatatgct gttccctttt 38400 ccttgaaatg gcccttaccc tcctttctgt tgggttttcc tatggaatat ccagtcagcc 38460 tttaggattc atcttgggtg tcccttcctg tatgtaggct ccctggccct ccaggattcc 38520 cccagtaaca gccctcatca tgctgccttt gcaaccattt gtttatttgt acctctcacc 38580 tgctagttgg gcaagttact cacttctctc aacctctgca tttttcttct ttataaatgg 38640 gaccaataat acctaccctg ccctggcgtg gatagattaa agaaaaaaaa tacatgcagc 38700 tgccattgag ggcctggccc acgtgtgatg ttcaataata ttatttctcc ttgttttcct 38760 tcccgtgcca gtgccacacc cccctgtccc agtgcactgg ggctgtggat cccttcaaag 38820 ctgagattgc ctgtctgtgg tctccagcgt taagcacagt cattagctca ggtgcgtact 38880 catgtgttcc acgagttcaa gcctcagccc tgtaaagttt gcctgccgtg tatctgatat 38940 atttctgcta aaacccatta ggcctttctt gctctgaaat gtcatcgtta gttgtgtgtc 39000 acttcagttt tgtaactggc caggccactg cgcccaggct gcttcctcgt catctggctg 39060 ctaaatgctt caaccttacc tgccttgcta tgcgtcccat cctgtatcag gtcagagctc 39120 ttgagtggtg aatacaaatt tcatttcagt tgacttttga ttcttgtggc aggcctctcg 39180 gcctactcta atttgattgc aacggacaca aaatgtgtcc aaacttgcag cttttcttct 39240 cttattttga tatcaccatc cacaaaggta agatatttta aagcaataac tacaaacttt 39300 ctgaaaatta tgaagaagtg ctgggtttta aatggaagtc atatagtgtg aactttgtgt 39360 aaagtccgta gggagttttc ttggaaatgg ctgggaacat tctttttgca cctttgaaga 39420 taaaggtagg tggaggagct cacagctctt gtgccatgtt gggcttgtca ctcttgttta 39480 tgtgccaaat tcttttgatt acaaaatttt aagtttaatg ctttaggtat tgttgggcaa 39540 gatctagatg tatctagtta aatgtaggtg atatgcaaac tatttatgat gtatttgatt 39600 taaattcatt aagatagagt gtctttacca ccattatagt ctggtccttt tccttctgtt 39660 ttaaatgtgt ttccattggc attttctaaa ctgactttgt tagcgtgtta atcatttggc 39720 actggtaatg attaatcttt tctttctttc tatttttttt cttttttttt tttgagacag 39780 agtcttgctc tgtcaccagg ctggagtgca gtggcgcagt ctcagctcac tgcaacctcc 39840 gcctcccagg ttcaagtgat tctcctgcct cagcctccca agtagctagg gactacaggc 39900 acgtgccacc acgcccagct aatttttgta tttttaatag agatggggtt tcaccatgtt 39960 ggccaggatg gtctcagcct cttgacctcg tgatccgccc acctcggcct cccaaagtgc 40020 tgggattaca ggcatgagcg actgcgccca gccgtgttca tctatttctg tgaaccgatg 40080 ctaggtgaag gtacagaggg ctttctagct tctgggtttg tttattctga aatgttattt 40140 taaatcttag cccaacaaat tgagcgaaaa gacttctaga tgttaaatat gatattcaaa 40200 aaatataaag acaaggtgat aaattagaat tggtgggaaa gagaaaaatc tgtcttctga 40260 tggtcacctg ccccagcaac actactcgtt tgagaagact tccatccttt accctcaaag 40320 tgttccatga ggttggatca gacatcattt agcaaagaaa gatgtaaata gatttctgta 40380 gggtggcatt attaagcata ttaagtggtt acaatacagt aaattagagg gagtagtaca 40440 gaagcataag cagtcaaaaa agtgaaagtc taacgttcgt aattattgtt ctggaggctt 40500 ttgtatcaca tataagttcc aggctgggta tgatggctca caccagtaat cccaacactt 40560 agaggccaag ccgcgtggat cgcttgagcc caggagttcg agaccaggct gggcaacata 40620 gtgaaaccta tctctacaaa aatacaaaaa ttagctgggg gtggtggcag cgcctgtagt 40680 ccaaaccact tgggagcctg aggtgagagg atcacctggg cccgggagat caaggctgcg 40740 gtgagccatg atcttgccat tgcactccag cctgagtgac agagagagac tctgtctcaa 40800 aaataaaaaa gttttgagtg tgaaaattca agctcaattc catttgttgg ttgtcttgag 40860 tgtctgatca catagaatat aaagatgttt tgatagttgg gacagtattc agctacctgc 40920 tatttaatac attatttcag aaaatattta caaagggggc tgggcacagt ggctcatgcc 40980 tgtaatccca gcactttggg aggccgaggt gggcggatta cctgaggtca ggtgttcaaa 41040 accagcctgg ccaaacatgg tgaaaccaca tctctactaa atatacaaaa aattagccgg 41100 gcgtggtggt gtgtgcctgt agtcccagct actcaggagg ctgaggcacg agaatcgctt 41160 gaacccggga ggcgtggggt tgcagtgagc cgagattgca caactgcact ccagcctggg 41220 tgacagagtg agactgcatc tataaaaaca acaacaaaaa agaaaatatt tgcaaaggac 41280 cttctgggtc caagaacctc atgtccaata caagggtgca cacgtgggtg agacacggca 41340 gctgctctcc agaagcccac agtggagggg tttcccttcg gtctcctttt attccaagca 41400 agtggcaaaa ctactttact cttaatacaa accacttcct tttatcacag gacgcttccc 41460 aagctctgca actgttgctc ctgaggaagg gagtggaact gataatctgt tcctccctat 41520 tgtgttcagt atggtttttt tttttttttt ccttttgctg gctttgtttt cctgtccctg 41580 tgatgattaa aattcactct gcaaattaga tcacctttcc cacgcagagt ccctttgact 41640 tctgttctag atatccatta catttttgta gtcttcggac acactgtgtg tgccgctttg 41700 ccctctgggt gacagcaggc tgtggctgcg gcgacagagc tgaggtgaat tctcacagac 41760 catcactggg ttactcctgg agtaagtaat tcccaagagc tccttctgtg cagatcgtta 41820 gaaatagata ttgaggccag gcgcggtggc tcatgcctgt aatcccagca ctttgggagg 41880 ctgaggcggg cagatcacga ggtcaagaga tcaagaccat cctggccaac atggtgaaac 41940 cttgtctcta ctaaaaatac aaaagtagcc gggcgtggtg gcgcacgccc gtagtcccag 42000 ctactcagga ggctgaggca ggagaatcac ttgaacccgt gaaacggaag ttgcggtgag 42060 ccgagatcac gccactgtac tccagcctgg tgacagagtg agactccatc tcaaaaaaaa 42120 gagaaagaaa gaaatagaca ttgaacacct gctacacagc agggattgtg ctagaagtat 42180 ggatgcaaag attaggtgaa tatgttccct agcctcacaa agcatacagt ctagtaggag 42240 agacagacac gtaaaaagtt tcaacagcac agataatcag ggctacacca gaattgggcc 42300 caagatgctg caggaatcta taggtgaatg ggtttcatga aggaagagct tcttctccca 42360 tttataactc attttagcct aatcttccaa acagtcacgc atctaagagc aggtgatgca 42420 gaaaataccc tcgtgttagt tatgaattac cgttggaatc cttccagtgt ttgcacctgc 42480 cctgtgctcg ggtaacataa aacagtgata taatttgatg tctacttcct cttgtatttg 42540 tctgttttta agtgttctac aattttcata tactctgttt catcgttctc aaaggaatat 42600 tttgattgat aaatgtttag ttagtaagac ctaaaaactg aatctcagta gtttgagctt 42660 atgatataca agatgcagct ctaacattta aattggaagg gaaatgtcaa aaagatacct 42720 gcactcttgt ttgttgcaac actgtttaca atagctaaga tttggaagca acctaagtgt 42780 ccatcaccag acaaatagat aaaggaaatg tgatatatat acacaatgga gtactattca 42840 gccataaaaa agaatgagat cctgtcattt acaacaacat gggtggaact ggagatcatt 42900 atgttaagtg aaataatcca ggcacagaaa gacaaacttc acatgttctc acttatttgt 42960 gggctctaaa aatcaaatca cttgaactca gagagattag aaggatggtt accagaggct 43020 gggaagggtg gtgaggggtt gggggcagtg aagatggtta acaggtacaa aaaactagaa 43080 agaatgaata agacccacta ggttttttgt tgttttgttg ttgttgttgt tttgagacag 43140 agtctcactc tgtcacccag gctggagtgc agtggcatga tctcggctca ctgcaacctc 43200 cacctcctgg gttcaagcga tcctcctgcc tcagcctccc aaatagctgg gattatgggc 43260 acgcgccacc acacctggct aatgtttgta tttttagtag ggaccgggtt tcaccaggtt 43320 gaacaggctg gtctcaaact cctgacctca agtgatccac tcgccttggc ctcccaaatg 43380 ctcagattac aggcgtgagc caccgcacct ggccactgtt tgatagcata atagggcgac 43440 tatagtcaat aataacttaa tggtatattt ttaaataact taaagagtat aattggattg 43500 ttgtaactga aaggatcaat gcttgagggc acagctaccc cattccccat gacgtgttta 43560 gttcacatta catgcctatg tcaaagcatc tcatgtaccc cataaatata tacactgagt 43620 atataccaca aatattttaa ataattttta aaataaaaaa ataaattgta agggaaagaa 43680 aattatgaat ttagaaatgt aaaaggtctc aggtaaggaa ggaatgagag gatcatgcag 43740 aacctcccat cattgctggg actggaacag aagccctacc ttttcccaac accctatcca 43800 cctgtccctc acctctcagc ttttgtgaga ctctgtctgt gctatgaaac tgaagatcta 43860 attcagtgct gtttgcattg tcttgcctcc tggaccagag gttgcagttg ttgagaaaag 43920 ggatggttgg ttatgccttg atccccccca gagcatttgg ggcataggac acgggaactg 43980 gccagcctgg ttcactcttc tcgattagct ggacagcggc atgtcatgtg ggtaatagga 44040 aggggtgggg acttccccgg gatattctgc tcctgatcag aagcgccagt gatgtggggg 44100 gagccccagc accagagcat gctgggaggg cgtgcagggt ggggcaggtg cccgtttggc 44160 ctctgctgtc tatctggggg atgcatccaa aggcaactgt tccttatctg ctcttgttgg 44220 gagcaaggaa gggccaattt gttcaatgat ccgtatacag ccagtccctc tggccagagt 44280 tcaagacagt attgcctcac tctatataga gattgtatct tggttagctc ttcattcata 44340 gcaagaccaa tgtttctgta aattaatcct ggtattgttt aaaagcaact aaaaatgatg 44400 aaattgtaaa actttgaaac tccctgaata taacgacaag caaactaaca ttgttttatt 44460 ggtcgatgct cctggccaga agagagaata ttagcaggga taaaaggcat aggccacatg 44520 cattttccac cccagtgctg agaacacgat gggcgaaaaa gggaggtggc cacagcccat 44580 ccatcacaca gtctctgccc atctacttgc tttttccttt tttttttttt ttttttttgt 44640 gacagagtct cgctttgtca cccagactgg agggcagtgg tgcaatctca gctcattgca 44700 acttctacct cccaggttca agcgattctc ctgcctcagc ctcccgagta gctgggatta 44760 caggcacctg ccaccaagcc cagctaattt gtttgtattt ttagtagaga cggggtttca 44820 ccatgttggc caggctggtt ttgaactcct gaccttaagt gatcagccca cctcagcctc 44880 ccaaagtgct gggattacag gtgcgagcca ccacgcctgg ccccagctac ctgttttctt 44940 tctttttttt tttttttttt ctttttttga gacaaagtct tgctcttgtc ccccaggctg 45000 gagtgcaatt gcatgatctc agctcactgc aacctccacc tcctgggttc aagcgattct 45060 cttgcctcag tctcctgagt agctgggatt acaggcgcct accaccacgc ccggctaatt 45120 tttgtatttt tagtagagac ggggtttcac cctgttggcc aggctggttt cgaactcctg 45180 accttaagtg atctgcccgc ctcagcctcc caaagtgctg ggattacagg tgtgagccac 45240 catgcccggc cccagctact tgctttctat tgggatgaac ctcatggtta atacagttag 45300 ttagtgactg caacttttga actttttgtt catagtgaaa aatattttaa gtaatgctta 45360 ccccattatg tttcttgtca tttgaaaaaa aatctccctt cagacagaat gcagaataaa 45420 atactacaga aaatctgtac agagtcccag cctgacttat gctagtaggt tacagagaaa 45480 gaaagtcttc taaaccctat gaaaggttaa cagttctctt atttttccct gtgtgctatt 45540 tgatgatttc cctgtgaact ttgatgattt attgccagaa ttccaaacat aatatgtgaa 45600 tttcacaaaa atggatgaaa tgtatctatt tttcattggt agaagaagcc aaaacatccc 45660 ttcctcaccg cactaaaagc tgttgtttac atgaagcaaa cctcaaatgt gaacatattt 45720 ttacgcaaat gcatttaatg ggtgaatatt tgctttggga cggtattctt tactctatct 45780 ggagagtctg gcgttccgta atcaccatgt gatgacggct gccctgacag tggctggtag 45840 cagcacatac ccccgagcct ctccgtggtg tgcgccgtgg gcaccatgtg accattttca 45900 gaaaggaaga cagttctgga agctaaaggt cacctagtca gcctcgttgg gtgattgatg 45960 actcagctgg gttcagggag gtggacccga ggcagagcct ctagaaggca gcggtgggca 46020 gggcggttca ggcaggtggc acctgggcaa aggtgcagac gtggaatcct gaaagcaatt 46080 ctcagcgctg ctgcgtttcc aggaggtaga agaacagtga caagtgcaca gtcgggtagg 46140 gacaaatgtg gaagggctgg gaacagtgtg ttcaggagac tgggcttcaa tctggaggtc 46200 tcaggaagtg gtttaggatg tttcagcgag agcatgatac agactaaccc aggaagaacc 46260 gctgctttgt cacttatacc cctatggaaa tgccgttcgc tttgctagtt gaaatagcct 46320 accattgtct gggactcacc cagttagatt tgtttggact ccacaaagta ttcttgacca 46380 tacaatcatg gtcgaggacc ccctacatga gctgccttca tggctacagg gagagcacac 46440 caaagtggat gtcacaccca gcacacatgc caccggcttg gccctgcgcc ccgcagcctg 46500 agccacactg gctgcctgtt cctggaatgt gccaacatgt ttcagtcctg gagcctttgc 46560 acttggtgtt ctcttcgctg gaacattctc ccccaagaca tttacacagc ttgccccctc 46620 attccctgag gttatctcct gccccctaat cagtgaggcc ttccctggcc tcaccccgga 46680 cactccacac gtgcattcat ttcgttgttc accatctgtg tcccagttac aagggaggct 46740 ccctgagagc agggatctga tttttgttag ttgttgttgt tgctgttttg aggtggagtc 46800 ttgctttgtc gcccaggctg gtgtgcagtg gtgcgacctc agctcaccgc aacctccgcc 46860 tcccatgttc aagcggttct cctgcctcaa cctcctgagt agctgggatt acaggtgcct 46920 gccaccatgc ccagctaatt tttgtatttt tagtagagac agagtttcat catgttggtc 46980 aagctgccct ccaactcctg acctcgtgat ctgcccacct gggcctccca aagtgctggg 47040 attacaggca tgagccactg cacccgaccc tgttttttgt ttggttttgg ttttggtttg 47100 gttttggttt ttttttgaga cacggtctca ctctgtcgcc ccggctagag tgtggtggca 47160 ccatctcggc tcactgcaac ctccacctcc caggttcaag tgattctcct gcctcagcct 47220 cctgagtagc tgggattaca ggcacatgcc accacaccca gctaattttt gtatttttag 47280 tagagatggg gttttgccat gttggccagt ctggtctcaa actcctgacc tcaagtgatc 47340 cgcccgcctc ggcctcccaa agtgctggga ttacaggtgt gagccactgt gcccggccca 47400 gggatctgtt tttgtctccg ctgtgtcccc agcacctcaa acatattgta cggagctgcg 47460 acagctgcgc agtcagtgat gactgagaga ttcctggccc cgtgggctat ggctccttca 47520 acagtttgtt gtttaaaggt tcttcacttt ctcagcgtgc tgatcaagag acaagcctgg 47580 aggagaggct cagtggtgct cctgtgtaga tgatgaattc aggtgtatct tggatggtaa 47640 atgacgttgc atttaaaacc aagcaagtgg ccaggcgcag tggctcacac ctgtgatcaa 47700 agcactttag aaggccgagg cgggcggatc acctgaagtc aggagtttga gaccagcctg 47760 gccagcatgg taaaaacccg tctctactaa taatacaaaa aaactagctg ggcgtggtgg 47820 cgggcacctg taatcccaac cactcagaag gctgaggcag gagaattgct tgaacccggg 47880 aggtggaggt tgcagtgagc tgagatcgca ccactgtact ccagcctggg cgacaagagc 47940 aagactctat ctcaaaaata aaaaaaatta aaaattaaaa tttaaaatta aaacaaacag 48000 ccggacgcag tggctcactc ctgtaatcgc agcactttgc gaggctgagg cgagcggaat 48060 acgagctcag gagatcgaga ccaccctggc taacacagtg aaacccgtct ctactaaaaa 48120 aaaaaaaata caaaaaatta gccaggcgtg gtggcaggcg cctgtagtcc cagctactca 48180 ggagactgag gcaggagaat ggtgtgaacc cgggaggcgg agcttgcagt gagccgagat 48240 tgtgcccctg cactccagcc tgggcaacag actgagactc tgtctcaaaa aaaaaaaaaa 48300 aagaataaat aaataaataa taaaaaataa aaacaaacaa gtgaacgttg ttatacgtca 48360 gtcttaccaa ttgttcctct ttcctcccag tagcttggag ctcggcggca caaccagcac 48420 catctggtcg cgatggtgga cacggaaagc ccactctgcc ccctctcccc actcgaggcc 48480 ggcgatctag agagcccgtt atctgaagag ttcctgcaag aaatgggaaa catccaagag 48540 atttcgcaat ccatcggcga ggatagttct ggaagctttg gctttacgga ataccagtat 48600 ttaggaagct gtcctggctc agatggctcg gtcatcacgg gtaagtgtgc cgtttcctag 48660 aaagttttat ttagaaatgt ttcttcctcc aagaaaactg ttctctcttt tttttttttt 48720 tttttttgag acggagtctc gctctgtcgc ccaggctgga gtgcaatggc tcgatctcgg 48780 ctcactgcag gctccacctc ctgggttcac accattctcc tgcctcagcc tcccgagtag 48840 ctgggactac aggtgcccgc caccacgccc agctaatttt tgtattttta atagagacgg 48900 ggtttcactg tgttagccag gatggtctcc atctcctgac ctcatgatct gcccgcctcg 48960 gcctcctaaa gtgctgggat tataggcgtg agccaccgcg cccggccgaa aactgttctc 49020 tttagctgga aaagaagtca cacttttttg caaagaaagc ttcagacgtg gtaaagcatg 49080 acctccagtg cccctgggcc ctggaaggcg cgtgtcacgg ctcacggtgc cccctcttgt 49140 gaaagccatg cacacatcaa acagtgcttg agattcagtc acggggaaca gctaaagtac 49200 acagacccta accccagcaa gcccgcgggg ggcagctaga catttttaag aggagacgtg 49260 tgcaagggtc tgcatagagg tactgttggt aagagggaag gatgggaaac aagctgtaca 49320 tgcgtcaaag ggaaacagat aaattgggat gcatttatac agtggtatat acttcatagc 49380 aatttaaaag aacagactag gctaggcgcg gtggctcacg cctataatcc cagcactttg 49440 gaaggccgag gcaagtggat cacttgaggt caggagcttg agaccagcct gaccaacatg 49500 gtgaggcccc atctatacaa aaaaaattta aaattaaaaa aaattagcca ggcatggtgg 49560 tgcatgcatg tggtcctagc tactcaggat gctgagggag gaggaccact tgagcccagg 49620 agctcgaggc tgccatgagc tatgactgcc actgcactcc agcctgggtg acagtgagac 49680 cctgtcttta aaaaaaaatt tttttaagca acattgaatg aaaataaaca agcttaatga 49740 atatttttat gatccaatta atgtaaaatc ttttattttt tattttttga gacagagttt 49800 tgctcttgtt gcccaggctg gagtgcagtg gtatgatctc agcccactac aacgtccatc 49860 ttccgagctc aagcagttct cctgcctcag cctccctagt agctaggatt acaggcaccc 49920 gtcaccatgc cgggctaatt tttgtatttt tagtagagat ggggtttcac cgtgttggcc 49980 aggctggtct caaactcctg acgtcaggtg gtccgcctgc ctcagcctcc caaagtgcag 50040 ggatcacagg catgagccac tgcacccggc ccaattaaaa tctttaacac taaacaatct 50100 agtacatcac tggtggaaac agacatacac ctattgcaaa gggcatctca gctttaagga 50160 ctcagtcacc tcctgagcaa gatggaggga gaactgggga ggggtcccat ggggactgta 50220 attctctcta ggttgtatat ttttaaaaga cttcagcagt gtgataaacc tgggtggtgt 50280 gtacatgggt attacagtca tgttgcttaa tgacagggac aggttgtgag aaatgcatcc 50340 ttaggtgatt tcatcattgt gtgaaagtca tagagtacac ttaaacccag atggtagagc 50400 ctgctgcaca ccgaggctct gcggtgcagc ctgttgctcc aaggcacgca cctgtacagc 50460 gtgttactgt actgaacggc gtaggcccct gtgacacaat ggtaagtatt tgtgcgtcta 50520 aacataccaa aacatatagt agaaaaggtt acagcaaaaa tacagtatta tcatcttatg 50580 ggaccatgat accacagttg aacttatggt ctattgttga ccaaaatgtc actgtgcagt 50640 gtgtgactat acagaaataa gctcagagaa attaagtaac ttggctgggc gcagtggctc 50700 acgcctgtaa tcccaacact ttgggaggct gaggcaggcg gatcacccga ggtcaggagt 50760 tcaagaccag tctggccaac atggcaaaac cccatctcta ctaaagaata caaaacatta 50820 gctgggagtg gtggcaggtg cctgtaatcc cagctactct actcaggagg ctgaggcagg 50880 gagaattgct tgaacccagg aggcagaggt tgcagtgagc agagatcatg ccactgtact 50940 ctagcttggg cgacagggtg agactccatc tcaaaaaaaa gttggggcgt ggtggctcat 51000 gcctgtaatc ccagcacttt gggaggctga ggcgggcgga tcacttgagg tcaggagtta 51060 aagaccagcc tggtcaacat ggtgaaaccc catctctact gaaaatacaa aaattagcca 51120 agcatggtgg tacacacctg taatgcctgg gcaacagagc aagattccgt ctcaaaaaaa 51180 aaaaaaaaaa aagtaagtaa cctgccacgg ttcatacagc cagaaagaca cagagccggg 51240 cctggacccc gcctctcagc ttgctctaga gggctattct ctgcatgctg gcatgatcgc 51300 gccttgtaaa aggtggcagt gttctcagct tagtcaatca ggaattgcaa gaggcaagtg 51360 agcccctgag gactctgggg ggcctttgtg accgagcagc tttgggagtg accctgacag 51420 acctttacag gtggtgcaag ttttgactcc ctttctcctg gcgcgttaag cagaggataa 51480 gcgctgtgga aggagtgaag gtgtagggag atcatggccc ccagagcagt ggggaagggg 51540 acagggaggc tggaggagag caaggaaaag gctccgtgtc aggtggcgcc ttgagtggcc 51600 tgggtaggtt gtcttgcagt gaacccgggt taatggcctt gacaatgacc gcattgtttc 51660 ctgagcactg caggctgccc acacacctca cacctcggct tgcctaagcc cagagcagcc 51720 ttgtgaggtc gttgttatgt ttatttaagg aaggaggaaa ggaggcaggt cccaggacat 51780 cctgacgtgc tggagatcac cagcccagaa cccagctctt aaccccacaa tgtgggacct 51840 ttcttcaccc atcacagaca caccccatgc tggttcaccg ttttcctata atgactattt 51900 gtgctattta ttagaaaaat cttttcctta tggatttgaa aagatttatc ttgcttttgt 51960 ttttcttttt tgcctttctt ttttaaggca ggcaggctcc cgcagcccca cccccagggt 52020 gaaaaatata gttcattgtc tagtaaaaga gttcagagat acactttttt ctttgggtaa 52080 gatatactct agagcttgtt ctgaaatatg gaatttgtgt gagctgcggg agtgggtggg 52140 tgtgtggctc tagctctgga aagttctttc ctggcagtgg ccaggagggc tgcccagccc 52200 cctcctgcct cctctggcag cttaaacaca ggacccctta ttctgtgctc tctcctgacc 52260 cctggtcctc atgcaggagg gaaccctgct cttctagggt ccttttctaa aagtagtgtc 52320 ttttaggtca ttgtcaagaa ctataatcta aaatgtattt ttaactcatc tggaaattct 52380 gacagaggta aggcttgaga atttcctgca tactagcctt gtggtctata taatccatta 52440 aaagccacat ttaacccaat tccacagact gaactgtgct tcccatctaa ataaattaaa 52500 agcaggccgg gcacggtggt cacgtgtgta atcccagcac tttgggaggt cgaggcgggt 52560 ggatcacctg aggtcaggag ttcgagacca acctggccaa catggtggaa cctcatctct 52620 actaaaaata aaaaaaatta gctgggcgtg gaggcgtgca cctcttaagc ttaaggacat 52680 atttcttatg atccaattaa tgtaaaatat tttatttttt atttattttt tgagacagag 52740 tttcactctt gctgcccagg ctggagtgca gtggtgcgat cttggcctgt aatcccggct 52800 actcaggagg ctgaggcagg acagtcgctt gaatccagga ggcggaggtt gcagtgagcc 52860 aagatcacac cactgcactc cagcctgggc aacagagtga gactctgtct ttaaataaat 52920 aaataaataa atagcgaggg ttcagggcag gagaaaaagg gttccaaatt tgttctgaac 52980 caattccaag gaactttatg gcacaaagaa aaaaaagggg aacttacaaa aagtgaccac 53040 actgaagcgt cctggtcacc catccctggt tttgaccacc agcctttaaa gtggcaagcg 53100 ggtgataacc catttcttat ttccccctca gcatttcctc actgttattc atacatgtgg 53160 tcatttgtac tcatctcaca attgttaaaa cctctttcct cccttccagg ttttactgaa 53220 ctgttactgc gaagtctgag agatgaggtc atttaagatt atttcttatt tgtaaattag 53280 atcgttcata tttgtaccta atctgatctt ttgggtaata ttcctagtta tgtagactgg 53340 tctctcagaa gagccggata ttaaatgcag tactttaaac tttacaccca ggagaccgga 53400 tgggtgaggc tggttcactc ggccaaagta ccattttatc tctgcttttt cttcccggct 53460 ttattgccat aattgacata caataaactg catgtattta aagtgtacaa tctgttgggt 53520 gtacacacac acgcatctgt gaaaccatca tcacactcaa aatagtgatg tagaaatttt 53580 gctccttagt tcgactaaat ctgggttctt gtgtcatgac caggaaaaat taggcacgtg 53640 gacacgttga agggtgagga gagcagtatt gggcgaaaag gaaaaaagaa aaaaactctc 53700 agcaaagcta gaggggatcc tgccaatgag ttcccagctc acagactgat tagcaggcca 53760 ccacacatga gctggaggcc aggctcctcc cgctgcgcaa ggtgagaact tcccgtggct 53820 ccaccccatt ctcccaatgc ccaggtgggt ccccgtccct tgcgggcctg tccagacaag 53880 ggaaccctgg gcaggttccc tcatctacac aaaagcacct gaggtaaaca cttgtggggc 53940 aggttgcaga ttctctgggg acgcccccct tctctgcctc ctgcatctat cagtagtgcc 54000 tctgtctgtc acccctaaag tttacttgtg ctgtttctaa ttcctctttc cccagccccg 54060 tgcctccctg cctccctccc ccagtaaacc atgaatccac tttctatcat tctaggttgc 54120 tttatatttc ctagaatttt atataaatgg aatcatacag cacgtactct ttctaggctg 54180 gcttctttca ctctgcagaa tggctgtgag actcatctgc attgcagcaa gcatcaatag 54240 ttcattcttc atccatcatg tggacatagc acagtttgct gattcacgca cctgttgatg 54300 agcatttagg ttgtttctag cttatggcta ttacaaataa agctgctatg aacattcacg 54360 tacaagtctc tgtacaaccc tctgctttca tttcttttga ataaatacct aggagtatga 54420 cggctggaac agatggcagg tgtttgtgta actttttaag aaactgccaa aatcttttcc 54480 agcatttcag aaaaatctta gaaaatgcta tactatgtta tattcccact ggcagtatat 54540 gggggagttc cagttcctcc ataccctcat caacatgagg catgatcagt ctttttaatt 54600 ttaaccatgt cagtaggtgt gtgatggtct ctcactgtgg tgatttttat ttgcacttcc 54660 ctggtgattt tgagcatctt ttcgtatgct tatttgccat atatcttctt tggtgatatt 54720 tctgttcaaa gcctttgctc attttttaat tgagttgctt ttctactatt cactattgaa 54780 cactatttat atattttgaa tacaaatact ttatcagaca tgtgatctac aaatattttc 54840 cccagtgtgt ggtttgtctt tcttttcttt ctactgatag tatcttaaaa aaaaaaaaga 54900 aaaaagattg ttttgtttgt tttgttttgt ttttgagata gggtctcaat ctattgccca 54960 ggctagagtg cagtggtgcg atcatggctt actgcagcct tgacctcttg ggctcaggaa 55020 accctccgac ctcagcctcc caagtagctg ggaccacagg tgtgtaccac catgcttggc 55080 taattttttt tttttagata cagagactcg ttatgttgcc aggggtggtc ttgaactcct 55140 ggactcaagc gaccctccca cttcggcctc ccaaagtgct gggattacag gtgtgagcca 55200 tcatgcccga ccagttctta attttgatga agtccaattt atcaatgtcc tttttttatg 55260 gatacttcat ttatttattt atttgagaga gggtctcacc ctgagcccag gctggagttc 55320 agtggcatga tctcagctca ctgcagcctc aacctcccag gcccaggtaa tcctcctact 55380 tcagcctccc aagtagctga gactacaggt acctgccacc atgcccgggt aagttttttg 55440 tatttatttg tagagacggg gtttcgccat gttgcccagg ttggtctcaa actcctgggc 55500 tcaagtgatc tgcccatctc agcctcccaa agtgttggga ttacaggcgt gagccaccat 55560 gcccagccat atatatatat atatatatat atatatatat atattttttt tttttttttt 55620 tttttttttt tttgagacag agtctcactc tgttgcccag gctggagtgc agtggtgcaa 55680 tcttagctca ctgcaacctc cttctctgag gttcaagtaa ttctcatgcc tcagcctctt 55740 tagtagctgg gattacaggc atgtgctacc aggcccggct aattaccagc cttatatttt 55800 tgaactctgt ttaaaacatt taggtgcata aacattcagg cttgttatat tctgttgatg 55860 aactgaacct tttattatta tgaaattgct gttgtaatcc gtggtaaaat tatttgttct 55920 gaacactact ttgtctgtta ttgatgtagc cactgcagct ttcttttgat tggtgttaac 55980 atggtatatc ttttcccatt ctttttcttt taactggttt gtgtctttat actatggttt 56040 gatttaaatc tattatctca caatttgttc tctttggtac atctttgttt tgttcccttt 56100 tcctcttttt atgccttctg ttgaattaat tgagtctttt ttgttttgtt tcatttaatt 56160 ttgttttttg agacggagtc tctctctgtc tccaggctgg agtgcaatgg cgctatctcg 56220 gctcactgca acctctgcct cctgggttca agcaattctc ctgcctcatc ctcctgagta 56280 gctaggatca gaggcatgca ccaccacgcc cggctaattt gtgtgtgtgt gtgtgtgtgt 56340 atttttacta gagacgggtt tcactatgtt ggtcaggctg gtctcaaact cgtgaccttg 56400 tgatctgcct gccttggcct cccaaaatgc tgggattata ggcgtgagcc accgcaccca 56460 gcctaattga gtcattttta agattccact ttatctcctt tgttggctta ttatttataa 56520 caccttctgg tgttatttta gtagttgctt tagggtttat agtgtatctc tctaatgtct 56580 cccagtctac cttccagtgg tatcattcta tcttacagat attataagaa ctttatgaca 56640 gtatactttc atttttccct tcatgcattt gtggtaatgt ttcacataat tttatttatt 56700 tacctacatt ataaatatta caatatgtta ttgttttaca tagacagccg gttatctttt 56760 taagatagta gtaagaaaaa ttttttacat ttacccacat aattaccttt tctagtgcta 56820 tatacctttg tataaatcca gatttccatc tgctatcatt ttccttctgc ctgaaagact 56880 tcctgtgata ttatctataa tatggctcta ctggtaacga attactagct tttgtatgtc 56940 tgaaaaagtc ttcatataac cttcattcta gaaagtatgt gattcaaagg gccgggcaca 57000 gtggttcacg cctgtaatcc gagcactttg ggaggccgag gcgggtggat cacctgaggt 57060 caggagttca agaccagcct gaccaataag gtgaaaccct gtctttacta aaaatacaaa 57120 aattagctgg gcatggtggc tcatgcctat agtccctgct acttgggagg ctgagacagg 57180 agaattgctt gaacccagga ggcagaggtt gcagtgagcc aagatcacgc cactgcacac 57240 cagcctgggt gacagagcaa gactccatcc ccctgcaaaa aaaaagaaaa agaaaaagaa 57300 aaaagtatgt gattctacat tggcaatttt tttttttttt ttttttttga gacagagtct 57360 cgctctatca cccaggctgg agggcggtgg tgccatcttg gctcactgca cgctccgcct 57420 cccaggttca caccattctc ctgccccagc ctcccaagta gctgagatta caggcaccca 57480 ccaccacacc cggctaattt ttttgtattt tttagtagag atggggtttc accatgttag 57540 ccaggatggt ctcaatctcc tgacctcatg atccgcccac ctcggcctcc caaagtgctg 57600 ggattacagg catgagccac cgagcctggc cacttttttt ctttaaatgc ttttaagatg 57660 ttcctactat cttcttgttt ttaattaatt aatttattat tattattatt attattatta 57720 ttattatttt tttttttttt ttttttttta gagacagggt cttgcgctga tgccgaggct 57780 ggagtgtgct agtgccatcg tagctcactg cagtctcaaa cacctggtct caagcaatcg 57840 tcctgcctca gcctcctgag gaactaggac tagaggtata tactaccatg cccagccaat 57900 tttaaaaatt ttttgtagag gtggagactc gctatgttga ccaggctcct ctcgaactcc 57960 tggcctcaag caatcctcct acctctgcct cccgaagtgt tgggattaca gggattacaa 58020 gtgtgagcca ctgtgccagt ccccactgtc ttctggcttg catcgtttct aaaagaaact 58080 tggtgtcatc cttatttttg tttctctaca tgttatatgt cctctttatc tggttgcttt 58140 aactttattt attaatttta gtttaatttt taattgacaa ataataattg tatttttatg 58200 gggcacaatg tgatgttttg gtctatgttt acattgtgga atgtgtaaat caagctagtg 58260 aacatatcca ccacctcaca cacttaccat tttttgtgtg tggtgagaac atgtaaaggc 58320 tgctccttga ggccaggccc aatcccagca ctttgggagg ccgaggcgag tggatcactt 58380 gaggtcagga gttcaagact agcctggcca acatggtgaa accccgtccc tactaaaaac 58440 acaaaaatca gccaggcgcg gtggtacacg cctatagtcc tagctacttg ggaggctgag 58500 gcaggagaat cacttgaacc caagaggcag aggctgcagt gagccaagat catgctactg 58560 cactccagcc tgggcaacag agcaagactc catctcaaaa aaaaaaaaaa aaagtctatt 58620 ccttgagcaa ttttgaaata cacaatacat cattgttaat tatggtcacc atagtgggta 58680 gtagatcact aaatcttatt cttcctgtct aactaaaact tttttccttt tgaccaacat 58740 ctccccattc cctccctcaa cctcagcccc tgataaccac cattccactc tctactgcta 58800 tgagtttgac ctttttagat ttcacatatg agatcacatg gtatttgtct ttctgtgcct 58860 ggcttctttt acttagcata ataccttcca gatttaccca tgttgttgca aatggaattt 58920 ccttcttttt taaggctgaa tagtattcgt gtgtgtgtgt gtgtgcgtgt gtgtgtgtgt 58980 gtgtatcaca ttttctttat ctcttcgttc attaatgatc atttaggatg attccacatc 59040 aggctactgt gtatagtgct gcagtaaaca tggaagtgta gacatctctt cagcatactg 59100 cttccaatct ctttggatat aaacccagaa gtgggattgc tggatcatat gtagtgctat 59160 ttttgttttt ttgaggaacc tccatactta ttttgcataa tgctattcta attcacaata 59220 ctaccaacag tggacatggg ttcttttttc tctacatgct tgccaaccac ttgttatctt 59280 ttatcttttt atatatctgg ctgcttctaa atttttttct ttcttaccaa ttctgaacca 59340 tttgatggtt tcttccttta tgctccttgt gcttgaggtt cattgagcat ctgggatcag 59400 tgcacttatt gttttcatca aattcagaag attaggccat tatttcttca aacttttttg 59460 tcgttctctg tctacctttg agagctccaa ttatacatac attaggccac ttgaagttgt 59520 cattacagtt cactaatgct aagttctttt tttaagtctt gtttctgtgt ttcattttgg 59580 acactttcta ttgctacatc ttcaaattta ctaatttttt cttctgcaat atctaatctg 59640 ctcctaatcc tatccagtgt attttccata ttagatattg tagttttcat aactagaagc 59700 atgatttggt tctgttttca cccatgtatc tatataacat gtccagtctt tcactcagct 59760 tcttaaacat ttagaatatg gtcagaataa ctttttttgc tgttttgttt tagagacagg 59820 gtctcacttt gttactcagg ctggagcgca gtggcatgat cacagctcac tgcagcccca 59880 acctcctcgt ctcaaggaat cctcccacct cagcctccta tgtagctggg accacaggta 59940 cacaccacca cacctggcta atttttaaat tttttgaaga gacgggtctc actttgttgc 60000 ccagactggt ctcaaactcc tgggttcaaa caatcctcca gccttggcct cccaacgtgt 60060 tgggattaca ggcatgagcc actgtaccca gcccagaata actttttaaa aatgtcttga 60120 ggccgaggtt gggaaataat ctgaggtcgg gagttcgaga ccagcctgac caacatggag 60180 aaaccccgtc tctacaaaaa atacaaaatt agccaggcac agtggcacat gcctgtaatc 60240 ccagctactt gggaggctga ggcaggagaa ttgcttgaac ccgggaggca gaggttgtgg 60300 tgagccgaga tcacaccatt ggactccagc ctgggcaaca agagcgaaac tccatctcaa 60360 aaaaaaaaaa aaaaaaaact cttagccaca atttctatca tctgtgtcac ttctgagtcc 60420 ctttctattc agttattttt ctccttgtca tgggtcatat ttttctgatt cttcatgtgt 60480 cctgtaattt tcttttcttt ttttttttgg agatggagtc ttactctctc acccaggctg 60540 tagtgcgatg gcacaatctt ggctcactgc aacctccacc tcctgggttc aagtgattct 60600 cctgcctcag cctcccaggt agctgggatt acaggtgctc accaccatgc ccagataatt 60660 ttttgtattt ttagcagaga cggggtttca ccatgatggc caagctggtt ttgaactctt 60720 gacctcaagt gatccgccca cctcggcctc ccaaagtgct aggattacag gcatgagcca 60780 ccgtgcctgg ccagttgttc tcattggatg tcatatgttg ggaactttat tgggtgatgg 60840 atatttttga tttcctataa atattcttga actttgttct gggatgcaat taagttactt 60900 ggaaaatctt tgatcctttc aggtcctgtt tctcagcttc attagatggg actatcacag 60960 tgtttgtttt agagataact ttgccccact gctgaggcaa aaccactttg agcttcacct 61020 gatgccccat gacttcagtg atcttccact gtgggaggcg agagcaggac tatatccagc 61080 tccatgtggg ccccaggcag cgttcactat catcatttca ggttgctact gaagtatccc 61140 tttttcaggc tctcagctgg cagagcaaat acatatatgt atacatacta acctatgtct 61200 atacaggaat ctatcggtat ttctgtctgt ggccatctgt agctgtatga agccaaacat 61260 gagtgtgtgc tgatgtctcc agccctcatc tgttaccaga tggatcgttc tagcctcctc 61320 cacttgccta cctgtcaatt caccattcct tgagttcatg gttcattttc agtatacctg 61380 cacagtggta tcagaactgt taacccacac cctgtgggaa aaaaactcca tcagctagag 61440 cacagtgttt acagccagat ccttttgcct ttagtcttac agattccaat cattccaaat 61500 tattcggtgc agcgcctttc cgcacctgca cccacttttt cccctgagat tgtttcctac 61560 attcgtagca cagttagatt gttttgttac attctgcatt tcaccctggg atcctccaac 61620 ctcctaagtt atttttgttt tatttgcaca cattaggttc aatctgaact ataaagttct 61680 gtgggttttc acaaatgcgt agtgtcatgt atccaccact acattttcct tctctctctt 61740 tcttgctttc tcgccttctt gtcttgctct gtcacccagg ctggagtgca gtggcacaat 61800 ctcggctcac tacaacctcc gtctcctggg ttcaagccat tctgctgcct cagcttcccg 61860 agtagctggg actacaggca cgcaccacca cccctggcta actttttgta tttttacaaa 61920 atacaaaaga cgatgtttca ctatgtgggc caggctggtc tcgaactcct gaccttgtga 61980 tccacctacc tcggcctccc aaagtgttgg gattacaggc gtgagccacc acacccggtc 62040 tctctccttc ctttcctttc ctctcctttc cttttctttc tttctctttc cctctcctct 62100 cttctcctct cctctccttt gatggaggtc tcactgtgac acccaggctg gagtacagtg 62160 gcagcataat ctcagctcac tgtagcctca gcctcccagg gctcaggtga tcctcccacc 62220 tcagcctccc aagtagctgg gattacaggt gcacaccgct gagcccagca aatttttgta 62280 ttttttgtaa agatagggtt tcaccatgtt gcccaggctg gtctcaaact cctgagctca 62340 agttatctgc cagcctcggc ctcccaaagt gctgggatga caggcatgag ctaccgtgcc 62400 cagaccactg ttagattttc atatgaatag tttcaccaca tcaaaaaacc ccatgcttca 62460 cctattcaac cctgcctctc ccacccccag ccagctcaga aatggttctt tttaccattg 62520 ctataatttt gccttttcca gaacgccatg aatttgaaat catatagtat gtagcctttt 62580 cagactgact tctttcatag caatatgcat ttaagagtca tccatgtctt tccatggctt 62640 gatatctcat ttctttttac actgaatgag ttcccactgt ctgtttgtac cacagtttgt 62700 atatctattc acctatctaa gggcatcttg gttgcttcca atttttggca attaataaag 62760 ctggccatgc acagtggctc acacctgtaa tcccagcatt ttgggaggcc aaggcgggca 62820 gatcacttga ggtcaggagt ttgagaccag cctggccaac atggtgaaac gctgtctcta 62880 ctaaaaatac aaaaattagc cgggcgtggt aatgggcacc tgtaatccca gctacttgga 62940 aggctgaggc aggagaatca cttgaacctg gaggcagagg ttgcagtgag ctgagatcgt 63000 gccactccac tccagcctgg gtgacagagt gagactctgt cccaaaaaga aaaagaataa 63060 actgctgtat acatgtgtag gttttgtgtg gacagaagtt ttcaaatcag ttggacaaat 63120 acctaagagt gtgattccat catacagtaa aactgctttg ctttgtcaga aactgccaga 63180 atgtcctcca agggggctgt ctcatgttgc attcccacca gcaatgaatg ggggttcctg 63240 ttgctccaca tcctcaccag atttgatgat gtcagttttg tggattttag tcatcctagt 63300 aggtgtgtgg tgacaccaca ttgttgttct cattctcagt gccccgatga catatcatgc 63360 tgagcattgt ttcatatgct tacttgccat ctgtatatcg tccttgctga agtgactgtt 63420 cagatgttca gatcttttgc ccattttctt tctttttttt tttttttttc cttttgatac 63480 ggagtcttgc tctgtcgcca ggctggagtg cagtggcaca atctcagctc actacaacct 63540 ttgcctcccg ggtccaagcg attcccctgc ctcagcctcc caagtagctg ggactacagg 63600 cacgcaccac catggcaagc taactttttc tttttttttt cttttctttt ttttttgaga 63660 tgaagtctcg ctctgtcacc caggctggag tgcattggtg cgatcttggc tcactgcaag 63720 ctccgcctcc tgggttcacg ccattctcct gcctcagcct cccgagtagc tgggactaca 63780 ggcgccccca ccacgcccgg ctaatttttt tgtgttttta gtagagacgg agtttcaccg 63840 tgttagccag gatggtcttg atgtcctgac ctcgtgatcc gcttgctccg gcctcccaaa 63900 gtgctgggat tacaggcgtg agccaccacg cctagccccc atttttcaat tgagttgttt 63960 gttttaagac ctctttgtat attaccacat gtgtattgaa aatattttct cccagtctgt 64020 ggcttgtctt taattttctt agcaatgtct tttgcagagc agaaggtttc attagctttc 64080 atagattcca acttatattt tctctttcat ggattgtgca tttggtgttg cccacacaga 64140 tttttatact gtattctggt gccatttact gagttaacaa ttgcggaaga actggaagaa 64200 aggaagcaaa caaaacgagt tctgcgtggc actgtcagtg cgggggcatg gggagtcctg 64260 cagggtgagg tatgggcggt atggcaaggc gcgggcccat agatgtgcag gtctggagat 64320 gtgtgcagcg gagatgtgcg ggcccgagat gtgcgggtcc gatgtgtggg tccggagatg 64380 tgcgcgtacc cagaggtgca gatcggaaat gtgggggtcc ggaggaaatg tgcggatcag 64440 gagaagtgcc agtcccgaga tgtgcggatc ggagatgtgg agggctagga gatgcgtggg 64500 tccggagatg cgcagatcag gagatgggcg aatcggagat gcgcgggtcc ggaaatgtgc 64560 agagcggaga tgtgtggatc aggagatgtt ggggggtcag gagatgcggg ggtccagaga 64620 tgtgggggtc cggagatgtg cgggtctgga gatgtgcaga gcagagcaaa gatgagctga 64680 tcggagatgc ccaggtccgg ggatccacgg gtccggagac gcgcgggtcc ggagatgcgt 64740 gggtccagag atgtgcgggt ccaaagatgt gcaaatctga agatgtgtgg atgggagatg 64800 tgcaggtccg gagatgcgcg gggcggagat gtgtggatcg gagatgctca gatcgaagat 64860 gtgggaatga ggagatgtgc ggggcgggat gtgtggatgg gagacgcgcg ggcccggaga 64920 tatgcggggc ggagatgtgc gggtccaggg atgtgtgatc tgaggtgtgt gggtccggag 64980 ctcggggtca gctcagcagc agtgagagcg agcatgctgg ctttgggagc acagcacaat 65040 ggcagctgta ggagtgcaag agggtgtgac ccagaggcag ggcccggccc cgcatgggtg 65100 ttctgaggtt tatgcctcag cactagaagc ctcgtatgcg aaatcacatc ctcatagacc 65160 cggttcagac acaggatagt gatgcctgga ctattcatcc gtctctcctc tttttcccca 65220 gacacgcttt caccagcttc gagcccctcc tcggtgactt atcctgtggt ccccggcagc 65280 gtggacgagt ctcccagtgg agcattgaac atcgaatgta gaatctgcgg ggacaaggcc 65340 tcaggctatc attacggagt ccacgcgtgt gaaggctgca aggtagaggg gagctggaac 65400 agggcctggt ggccgccacc atcaactact tatggtcact tttatagcaa atggcagtca 65460 ttactgagag attgcagaaa gtcccggata agaaactgac ttcaggccag gcgcggtatc 65520 tcatgcctat aattccagca ctttgggaga ccgagatggg tggatcacct gagatcagga 65580 gttcgatacc agcctggcca acatgatgaa accctgtctc tactaaaaat accaaaaaaa 65640 attagccagg cgtggtggtg ggcgcctgta agcccagcta ctcgagaggc aaagacagga 65700 gaattgcttg aacccaggag ccagaggttg cagtgagcca agattgcgcc actgcactcc 65760 agcctgggca acaagagtga gactccatct taaaaaaaaa gaaagaaaaa aagaaaaaga 65820 aaaagaaact gacctcagtg atagattagc ctctctttat agcacagaac ccctgagagc 65880 gtaagccctg ttgtgaactg cgtatttgag gaatctagct tgtacgcccc ttatgagaat 65940 ctaatacttg atgttccaag gtggaacact ttcatcctga aactatccct ccccaccccc 66000 atctgtggaa aaattgtctt ccatgaaacc ggtccctggt ggcaaaaagg ttggggattg 66060 ctgctttaga gagtctagga caaatggttc ctctgtgctt tgtaaatact tagagaagtg 66120 cattctttaa aagaaaataa gtcacattgg accgggtgca gtggctcacg cctataatct 66180 cagcactttg ggaggccgag gcggctggat cacctgaggt caggagttca agaccagcct 66240 ggccaacatg gtgaaaccct gtctctacta aaaatacaaa aattagccag gtgtggtggt 66300 gggtgcctgt aatcccagct acttgggagg ctgaagcagg agaattgctt gaactcagga 66360 ggcggaggtt gcagtgagct gagatcgagc catttcactc cagcctaggc gacaagagta 66420 aaacttcatc tcaaaaaaaa aaaaaagaga gaaaagaaaa taagccacat taagaacatc 66480 acttcattcg aatacaagac agagagctgt taccgttgat ctctggagcc tccctgaagg 66540 ccaggtgggg caggtgttct catgctcctg ccagggaaat tggccatcag agacacagag 66600 tatcttgctt agggtcccac agcccccagc agcagggact ggaaccagag actggctgct 66660 cctgctcccc agcagttcct tcctgcacat caggggcttc tccacctgat tcaagcgaca 66720 ggaaccccct gtgcatcttc atcctcctgc tggctcagcc tgccctaaac agatgtgacc 66780 tgggccagga gtgcatgaag gcaggccctg ttgtcctgca tgctgccagc tggactggtg 66840 gcccttccgt gtttgtcagc gtggtgatga ggagagctcc tgtagcagcg tccctttagg 66900 gttgcacaga cgtgctcaag tctggcgcct tatgtacgtg atatgtggga gatcatcatc 66960 tgaatgtttg gtttgaatca gaaatccctt ctcacggtgc acgctgcagg tgttcactaa 67020 cttggaaaat gccaccgcct ttctggcaca atgtaccatc ttggaacacc agcattctgc 67080 cctgagccag gcctggcctc agaggcctgg gccacaggga gaacctcaca gccaggacac 67140 tgtggcactc tgctgtctag aagcctgtct ccccaccctt cccattctaa ccccatgcgt 67200 tcctcagcct ccccactgtg caagcctagg taaggacatt atgaagacgt cagcctgcct 67260 ctcacattcc cctgcacact gctgtccctc tcccgcgggc caagcagacc cactgtggca 67320 aaaatataga agaatgactt aaaagcaaag agaaaaaaga acccaaagca aaaatgaact 67380 ccttcgcatg ttttctaacc atataccttt gaaaaagctc cttataaagt ggccttttcc 67440 ttagggccat gattaattat tcatttagtt ttgtttttta tggactattt agtaacattg 67500 tttcttgctg ggtagagttt aagatgcttt tacaaagcaa gaaaattgtt tacaaacagc 67560 tggcttcctt ttattataat ttttgtcttt gagggagtta atatactctt acaaaaattc 67620 ttagaaagtc tttagtcaca aatatggaaa tgtcacaatg ctggggatag ttacattcat 67680 atacattgta acaaggctga gtaactcttt ggaaaactat aattgtgttt tcccaagtca 67740 gatgagggca ttttgaaatg acttcgaatg ctgcctcatt ttattgtttt tcacattaaa 67800 tgtaacgaca tttaaagttc tgtatttgtc ctaatcattc cagacttctt agaagaacta 67860 tttctttctt tttttttttt tttttttttt tttttttgag atggagtctc actctgtcgc 67920 gcaggctgga gtgcagtggc acaatctcag ctcactgcaa cctccgcctc ctgggttcaa 67980 gtgattgtcc tacctcagcc tcctgagtag ctgggactac agacttacat caccatgccc 68040 ggctaatttt tgtattttta gtagagacag ggttgcacca tgttggctag gctggtctcg 68100 aactcctgac ctcaggtgat ccacccgcct cagcctccta aagtgctggg attacaggca 68160 tgatcaccat gcctggcctg gaataacttt tctctaaatt ttgttcattt aaaaagaaac 68220 aataaatgag caacaaaaaa ggtgagtaaa gcaagtgcgc tggtttctca gtggcccagg 68280 tctttaaatc cactgtgtat taccctcaca gggcttcttt cggcgaacga ttcgactcaa 68340 gctggtgtat gacaagtgcg accgcagctg caagatccag aaaaagaaca gaaacaaatg 68400 ccagtattgt cgatttcaca agtgcctttc tgtcgggatg tcacacaacg gtaggtaagg 68460 tggccctgca cattttccca gttcgttcct cagttcccct tccttgctcc aagggaacag 68520 atcaagctat ggatgaatgt gcttcaacat ttcacaccca agtcattttg taatcagagt 68580 ggcctaagaa aataaaagtc gcccaggcgc ggtggttcac gcctgtaatc ccagcacttt 68640 gggaggctga ggtgggtgga tcacctcagg tcaggagttt gagaccagcc tggccaatat 68700 ggtgaaaccc cgtctctact aagaatgcaa aaattagctg ggtgtggtgg cacatgcctg 68760 tagtcccagc tactcgggag gctgaggcag aagaatcgct tgaacccggg aggcggaggt 68820 tgcagtgagc tgagattgcg ccactgcact ccagcctggg cgacagaggg agattccgtc 68880 tcacaaaaaa aaaaaaaaga aaaagaaaga aagaaagaaa ataaaagtct cccaggtgcg 68940 gtggttcaca cctgtaatcc cagcactttg gaggccgagg cgggtggatc acttgaggtc 69000 aggagtttga gaccagcctg gcgaacatgg caaaaccccg tctctaataa aaatacaaaa 69060 attagctggg catggtagtg cacacctgta atcccagcta cttgggagga tgagacagga 69120 gaatagcttg aacccgggag gcggaggttg cagtgagctg agatcgcacc actgcactcc 69180 agcctgggcg acagagtgcg actccgtctc aaaaaaaaag aaaaaaaaag aaaaagtctc 69240 aaatagctga gattcagtgg tgcattggac tcgctgttag aaacttcagt ggtaagactt 69300 tgatacagaa tcgaaaaacc aagtggaagg caccaaaatg acagaatgtt cacctcgtcc 69360 ataggaaggg tgtaccacct caaacatctc accacgttat gaatttcctt ctagccaatc 69420 atttaatagt ttcagaacat gctaattgtg atgtgaatgt aagtcgttca taagagttgc 69480 atgtctacct tctggaaaaa gaagcagtta ttatataaac tcatcccgaa gccccgttca 69540 cctccttcac tcaaaggttg atgatgcacc tgatagtggt gtgcacccta ctaatgagac 69600 gaacgatggt gtcaccttca gcctgcacct gttaacgatg gtgtcacctt cagcctgcac 69660 ctgtttaaac atctacagtg tatggagttt gagtttttca tctctccata gtggaaagcc 69720 gaatagtaat gaaggatggg tctgaactgc ctgtgaattt tcattcctgg tttaaagtcc 69780 tgggggagcc cctcgtccag ccctgtccgc gcagtcatga cctcactgct catgcctgtg 69840 tttccccctc caaaccctag cgattcgttt tggacgaatg ccaagatctg agaaagcaaa 69900 actgaaagca gaaattctta cctgtgaaca tgacatagaa gattctgaaa ctgcagatct 69960 caaatctctg gccaagagaa tctacgaggc ctacttgaag aacttcaaca tgaacaaggt 70020 caaagcccgg gtcatcctct caggaaaggc cagtaacaat ccagtaggtg tttgcggctg 70080 ttctgggttc tcttggcaac atggaaccag tgtcgtagag gacgattaag gacacatgtg 70140 ttgaatgttg agaaaattat atttatccca cagttaagca aaggacagcg aagatggaaa 70200 cagttcattc tgagactctg agctgtagct taacaacaac tcctttcttc ttgcttggag 70260 ccacctcaaa gctcttagca actaagttat tatactggct atgtaattaa tacacttaaa 70320 aaaaacctta atagcttacc aagtactaag atgatttctt aggagcattt tttcttaaat 70380 agagataggt tcttgctctg ttgcccaggc tggaatgcag tggtgcaatc atagttcact 70440 gcagccttga actcctgggc tcaagcaatc ctcctgcctc agcctcccaa ggagctggga 70500 ctacaggtgt gcaccaccac acctggctat gtttgatgtt gttgttgttt tgttttgttt 70560 ttgttttttg gtagagatga gatgtttccc aggctggtct caaactcctg gcctcaagtg 70620 atcttcccac ctcggcctcc caaagcactg gcattacagg tgtgagtcat ggcacccagc 70680 attaactgga tttaaaaaaa aaaaaactga ccaggcaaga tgggtcatgc ctgtaatcct 70740 ggcactctgg ggaggccaag gtgggcagat tgcttgagtc caggagtttg ataccagcct 70800 ggccaacatg gagaaacccc aactctacta aagatacaaa aattagctga gcagggtggc 70860 acacacctgt aattccagct acttgggtgg cttaggcatg agaattgctt caacccggga 70920 ggcagaggtt acagcaagct gagatcatgc cactgcactc cagcctgggt gacagatcga 70980 gaccctatct caaaaaaaaa atagaataat aaaataaatc cctactttga ggtgtattag 71040 tctgctataa agaaatccct gagacctggt aatttataaa gaaaagaggt ttaattggct 71100 cgtggcccac aaggctgtac aggaagcttc tgcttctggg gaggcctcag ggaatttgac 71160 tcatagcaga aggtgaagtg ggagtaggcg tcttgcatgg caggagcaaa aacaagagac 71220 acacactttt cacccatcag atcttgtgag aacgctatca ctagagtagc accaagagga 71280 tggtgctaaa ccattcatga aggatcaccc ccatgatcca gtccctcccg ccaggcctca 71340 cctccaccac tggggattac agttcaccat gagatttggg tggggacaca gagccaaacc 71400 atatcataag gctagaaaag gaaaccactt acttcccact caaaatgtgc tcttggtcct 71460 ttctcctaaa actactccct ccctctcaga caaacatgcc tacattcttt ttccgccttc 71520 agtgaaaaga cagtgacatc ttggggctta gaaagggcca cttgtaagcc aggcgtggtg 71580 gctcacgcct gtcatcccag cactttggga ggccaagaca ggcggatcac gaggtcagga 71640 gatcaagacc atcctggcta acatggtgaa acaccatctc cactaaaaat acaaaaaatt 71700 agccgggcgt ggtggcgggc gcctgtagtc tcagctactt gggaagctga ggcaggagaa 71760 tggcgtgaac ccaggaggca gagcttgcag tgagccgaga tcgtgccact gcacttccag 71820 cctgggcgac aaagccagct gtgtctgggc gcggtggctc atgtctgtaa tcccagcact 71880 ttgggaggct gaggtgggtg gatcacttga ggtcaggagt ttgagaccac cctggccaac 71940 atggtgaaac cccatctcta ttaaaaatac aaaaaattag ctgggcatgg tagcggttgc 72000 ctgtaatccc agctacttgg gaggctgagg caggagaatt gcttgaacct gggagctgga 72060 ggttgcagtg agctgagatc gcaccactgc actccagctt gggcaacaga gtgagactct 72120 gtctcaaaaa aaaaaagaaa ggaaaagaaa ggaccacttg ttatagaaag cctgtctttt 72180 aaggtagctc tggacctttt cagaggcagc caaattgccc ctcatggttc gtcccccaca 72240 tccccgcctg cctggcctaa gtcctccttc cccctcccca acagttaaat aagtctttgt 72300 ctccattaca aaacaaatct cagagctacc ttcaaagaag agccagccct cagttggtga 72360 atgaagatac tttgacattt tcctatgagc atggtgaaac aggtttaatt tgtattaaat 72420 agcttgaagc aatccttatt gggaattaca aggtggaatt ttagtcacag gaaaataaag 72480 catttcacaa gctacttact ttcatgaaca aaccaaacct cttctttact gagtccttta 72540 attcttcagt gaattctcca attaaatagg ccgagacatt ttagaagttt ccagcagaca 72600 cccacactag gcagctccag aggcttgtcc caattagaac tttcctggat tacgagagtg 72660 aaagaaaagg taacttttag cttcgagtct ctatcctgga tatgattagt acagcccaaa 72720 attgggatgg ctaaaacttt tgtttgccag cttatatttc tcccttggat ttcagaattg 72780 aaagcaggct gggcacagtg gctcacactg taatcccagc actttgggag gctgaggcgg 72840 gaggatcact tgaggcaatc caagagtttg agaccaggca acacaaggag acctcgtctc 72900 tacaaaaaat gattttttaa aaaactagct gggcatggtg gcatgtgcct gtggtcccag 72960 gtacttggga agctgagatg ggaggatggc ttgagcccag gagttcaaaa ccaacctggg 73020 caacatggca agaccacatc tctacaaaaa ataaaaacat tatccaggca tggtggcaca 73080 tgcctatagt ccccgcgact tgggaggttg aggaggatgc cttgaggcca ggagttcaag 73140 gctgcagcga gccacgatcg cgccactgca ctccagccta ggcgacaaag cgagactctc 73200 taaaaaaaat tcgaagcaga gttaagttgt ctttcttcct aacaacctgc ccccaccatg 73260 gggtgcgaat gggactcctg gagtcctcct gcacctcccc ttggagacca ccaagctcta 73320 ggaaccccat caccctcagc tgagggtcac atgcagcaac tagcaggcgg gaatctgttt 73380 gcattttggc cttaaagaaa taaataatag gccaggcgcg gtggctcatg cctgtaatcc 73440 cagcactttg ggaggctgag gcaggtggat cacctgaggt caggagttgg agaccagcct 73500 gaccaatatg gtgaaacccc gtctctacta aaaatacaaa aattagctag gcatggtcgt 73560 gggcacctgt aatcccaact acccaggagg ctgaggcagg agaattgctt gaacctggaa 73620 ggcagaggtt gcagtgagcc gagatcacac cactgcactc cagcctgggt gacagagcga 73680 gactccatct caaaaaaaaa aaaaaaagag ggccaggcgt ggttgctcat gcttatgcct 73740 gtaatcccag cactgtggga ggcagaggag ggcggattac ctgagctcag gagttcgaga 73800 ccagcctggg caacatggta aaaccccatc tctactaaaa tacaaaaaat tagccgggca 73860 tggcagtgtg cgcctgtagt cccatctatt cgggaggctg aggcaggaga atggcgtgaa 73920 cctgggaggt ggaggttgca gggagccgag atcacaccgg tgcactccag cctgggtgac 73980 agagtgagac tccatctcaa aaaaaaaaaa aaagaaagaa atgatagatg aatagtttag 74040 gattggggtt cacaatttgg ttttctgtag aaaaagagaa ccgggcactc ttccgagagt 74100 cagatgccct cttccaccca cacccacaaa gccagagcac cgcaggtacc agttttcaag 74160 gcaacctcca accatcatgt gactctttgt gtttgatcac actgtttgct ccaagccagg 74220 gttgcgtccc accccatgtc cttgtctgcg cacgggacgc tggaggcacg gccccctcct 74280 ccctgcctag cctgctgacg ggctttccag agctggctcc ttcaggtgca ggataccctc 74340 tctgcttagt ctgggaaaag gccccgttgg caggatgccc accaccaggc cacactgcct 74400 gaatctattg gcagagctct ggttttgtgg ccaaggtggg tagtggaaga ccatagcctg 74460 tgtcccttac acatctcaga aagcaacccc atctgtgggc aagaaatctg ttagggagac 74520 caagcagcgg cctggaaaca ccttgatctc tgcccagtgg cccacatgcg gtcgccgttt 74580 catcagtttc cagcctgggt gacctcacag ccccagccac gccccacaga gcctcaggaa 74640 ggcacactga cctcagggcc ggcggctgac ttcatttctg tttggggatg agaggcggca 74700 cagtaaactg tccaggccag taaactaatg gattcatacg aaccgtaatg aacgtgggct 74760 gtgtgctggg gaaggcaggc tcgcctcctc cctgcagggg ctgctggggt gaaagcaacc 74820 ctgaaatgtt caaagccttg atggggaagc acgggggatg gatagatttt aatttcaaag 74880 cagccctctg gtttgctata agcgggggac tgaatttctc tttgcagtgg ccaatgcctt 74940 tcttctgtca agatcagctc gtggccttca gatcagatga cgcaaagccc catggctgag 75000 ctggaacagg ctagaatgct gggggggggc ctgaaaccgg tgggggagtt gtgggaggcc 75060 tagaatcagc caggaggctt gggtcggggt tggaaccggc cagggtgcac ggaggaggct 75120 gtgggggcag ggggaggccg ctgcatggag ccgcatagat gccattgctt gaggaaaggt 75180 gggctttagc tgagggaagg agtgaggggt ggatggagaa tgtctgtgtc catctggaca 75240 ctgggactgt ttgagcccct gagatttcag aaccgtgggc cagaaaatgg tcagggccct 75300 tggtgatggg gaagggcgcc tctggggaac tcactgcccc ttgatttgag ggtaacaggg 75360 atggaagcag agtcaggggg ctgagggagg caataaaaat gggtgctttt caacagtgtc 75420 taaaaacata agatgttgac ctgtcagggg ttgagaatgt cgtcagaaga ctttggagga 75480 agcaacagaa aatgagactg aggggcttgg gcagagtcag tgccttctgt gtgatgcacg 75540 ctcatgcaca aatgcacgca catacccaca ctcacacatc cgtgcacaca cgggtacaca 75600 cacatacacg tgcacccaca tgcatgctca cacacatgca cccacagtca cacatccatg 75660 catgcatgtg tacacaaaca cacccacaca tacacatgca cccacacgtg tacacagatg 75720 cacctccacc cccatacatg cacatggaca cacacatgca cccacacgca cacaagcatc 75780 catgctcaca tgggtacaca ctcacacatc catgcatgca cgtgtaaaca cacacacccc 75840 cacacataca cgtgcaccca cacatgcaca cagacgcacc tccaccccca cacacgcaca 75900 cacacacatg cacccacaca tggatacacg cacactcaca catgtaccca cacctgtgtg 75960 tacacacaca catgcatgct cacacacatg cacccaggca cacacaaatc cacattcacc 76020 catacagtca cacacatgca tacacacaca tacaaacaca tgcattcaca cagatgcata 76080 cacacacaca cttacaaact acacatgtgc ttatacatgc tcacatgcat gtatatgcac 76140 acacataccc tcaccttatg cacacatgta cccacacacg tacccacaca tatacaagca 76200 tgcacacata tatatatata cacatgctca cacgcatacc cacactcaca tgtgtgcaca 76260 tatgctcaca cacacgtgca cacacatgct cacacacaca cttactgttg ctcaggctta 76320 gctgctttgg gcttaagaag caaactgcac cttccaaaaa atgagtgtgg tgttcagtta 76380 aacaaccaaa taattcttta gcactgaata tgtggacttt agaaattcaa actataaggt 76440 gataataacg ttgtcctgct actttttaat ctaacaaaca tatcagaact gacactcagt 76500 tcaaatgaag aaagtaggaa ttgggcgtgc cgtgttattt tttcaaagat tctcctattg 76560 ctccaaattg ttggggatta tcttaaagtc tttgaatagc ttcagttatg gaagatttta 76620 ccctctgaga atagaactga attttagaca aaccatgagt ccattgtagc tagactggca 76680 tgcaagttgg gattaaacag agtaaaacgt cttgtttaaa aaaataagaa aggccggctt 76740 gggcaacata gtgagacctc ctctatgaaa agttagctgg gcatggtggt gtgcgcctgt 76800 ggttccagcc gctcaggagg ccgaggcagg aggatggagg tcaagactgc agtggactgt 76860 ggttgcgcca ctgtactcca gcctgggtga cacagcaaga ccccgtctca aaaaaagaaa 76920 acagaaaaaa gaaaaaaaaa gttgagcaag gagactaatt tgtgacatgc agctgaacat 76980 ggtttttaag accagttttg aaagaggaat tccaacatta ttcttaacat ttcagaagcc 77040 tgggcataag ggtgacctcc agggtgccgt gttataacag gactgctcct ttcaacagct 77100 atgaccttat accatgtctt ggggtgttgc ctgccgtgtg acagtccaat attataccta 77160 ctacttaagt tttctttaga ttaaaaaatg tgcttcatat tttatgccat ttctacaaat 77220 gtatagtaaa acataaccaa gagagcttat taaataattt catccaaagc agttctacca 77280 gtgcttcaca tttatttttt atttatttat ttatttttga gactgagtct cactctcttg 77340 cccaggctgg agtgcagtgg cgcaatctca gctcactgca acctccccct cctgggttca 77400 agcgattctc ctgcctcagc ctcctaagta gctgggatta caggtgccag ccaccacacc 77460 cgactaattt ttgtattttt agtagacacg ggcttttgcc atgttggccg ggctggtctc 77520 gaaatcctga cctcaggtca tccacctacc ttggcctccc aaagtgctgg cattgcgggc 77580 atgagctact gcgcctggtc cacatttaat tttttgcaaa aagatgacag ctgctaacag 77640 agatgaattc tcatgagtga tatcattgag cttcgtaggc cacatgagtg tgtgccggga 77700 ccagtgtggc agcaagcggg gcgttctgct ctcggcatgg agtgattggg gaaaatctag 77760 gcagcttcct gcctcacgct gtttaaaacc tttataatgt gctttatttc atttatttga 77820 aatgactgcc tgtcgtgtca gatatattca tagtcaagct tgagtataaa aggcatattc 77880 caaagttaaa tataagctgc tgcatagatt tttttgtaaa atgatctcac caagaatgtt 77940 tatccataaa gtttagcgaa tttgcaagtg tgtttttcaa cagcatttct ctttagcttt 78000 aataaacatt ggtttcttca tggtaccact cattttgaat tcagtggtct ccagttctcc 78060 ctgctaaatg aggcccactt tctaaaacca aagtgataat tttataaaaa tgaaatgaga 78120 tatttgttac cacagaagtc ctcatttacg agagtacatc cccatagaac tagtccacgg 78180 tgagcctcag gggcatgcaa gctgtttaac gatgccccca gcctagaaag gcccaggctt 78240 gggtgttcat gctccgctgt tgccttcttg aaattcataa tcatctttga acaaggggtc 78300 ccgcagtgtg tggtggctca cgcctgtaat cccaacactc tgggaggctg aagcgggtgg 78360 atcacctgag gtcgggagtt tgagaccagc ctgaccaaca tggtgaaacc ccatctctac 78420 taaaaataca gaaattaacc aggcgtggtt ggtgggtgcc tgtaatccca gctactcagg 78480 aggctgaggc aggagaatca ctcaaacctg ggaggtggag gttgcagtga gtcgagatca 78540 cgccactgca ctccagcctg ggcaacagag cgagactccg tctcaaaaaa gaaaaaccaa 78600 ggggtcccac atttgcattt ttgctctggg tcctgtaaat tacgtagcca ggcctgcatt 78660 tgtcctggga gatgctctac caaaaaacaa taaataacac caagcattct gtaatcaaac 78720 actgtaggaa cccctgctta tcctagcctc attctcattc tggaagactg cacatttatc 78780 atgttaaaga ctcagctagg gaggcccaac ttcattcaac tcagtgtttc ttattttttt 78840 aaaacagaac tcatttttta aaaaaattat tggctgggcg tggtggctca cgcctgtaat 78900 cccagcactt tgggaggctg aggtgggcgg atcacgaggt caggagatcg agaccatcct 78960 ggctaacacg gtgaaacccc gtctctacta aaaatacaaa aaaaaatgag ctgggcatgg 79020 tggcgggcgc ctgtagtccc agctactggg gaggctgagg caggagaatg gcatgaaccc 79080 gggaggcgga gcttgctgtg agccaagatc acgccactgc actccagcct gggcaacaga 79140 gcgagactcc atctcaaaaa aaaaaaaaaa ttatttaaca cctttatttc tgctgaatgt 79200 actttagaaa gattgagtga tttgaataaa gtgacggtgg cctaagagtc tattttctgg 79260 aattgaggga atactgccat cgatccttga aaaatattta tttagttcct cctagaggcc 79320 gggcacagtg gctcacgcat gtaatcctgg cctgcacttt ggaaggctga ggtggacaga 79380 ttgcctgagc tcaggagttc aagaccagcc tgggtaacaa ggtgaaaccc gtctctacta 79440 aaatacaaaa aattagctgg gcgtggtgat gtgtgcttgt aatcccagct actcgggagg 79500 ctgaggcagg agaattgctt gaactcagga ggcggaggca gaggttgcaa tgagctggga 79560 ttgcaccact gcactccagc ctgggcaaca gagcaagact ctgtctcaaa aaaaaaaaaa 79620 aattatctag cccctcctag aaatgttaat tccttaaatc tgagcttcag ctttctgtga 79680 agcagaatta tctccaaact ttaacaaaca atggtcagaa ctgtttttaa ggtcttggag 79740 agagatcatt ttcagtcttt attaatcgga cttgagatta tttagaaact tggctctgaa 79800 tattgtattc agaatgtttt cactcatttg tgagtaattt tttaaatatc ccctttcctc 79860 agatgcagaa tcagggcttt ttgtccagca ttatgttgca agtcctggtt ctgttgaaac 79920 attccatacc atctgtgtga tggttatcgg cacctccacc ggtgccctga agacagtttt 79980 gtgctgtgag tccagaaaca ggaaacactt caggctgtgt gtcagaagca ttgtcagtgg 80040 ttgtgttttg cccactggca gggggcattc tttaaatcct gggatgcttc tgcgctttgg 80100 gctccactgt tccagcagtg attagaaata acgctgtagg ccgggcgcgg tggctcaccc 80160 ctgtaatccc agcactttgg gaggctgagg tgggcagatt acctgaggtc aggagtgcga 80220 caccagcctg accaacatgg tgtaaccccg tctctactaa aaatacaaaa ttagctgggc 80280 gtggtggcgc atgcctgtaa tcccagctac tcagaaggct gaggcgggag aatcgcttga 80340 acctgggagg ccgaggttgc agtgagccga gattgtgcca ttgcactcca gcctgggcaa 80400 caagagcaaa actctgtctc aaaaaaaaaa gaaataacac cttagcccac tgcattattg 80460 acctgtgtct gcatgagctg tggaccacat tataatcaga gagatctctc agatgttgtc 80520 actttcctgc tctacccgca gatgtaaatt tcagccaaca gcagtgtttg tgctcatttt 80580 ccccggctct cccacacatg taatcccttc tgagcatgtt ggcttcaaat aatatggcca 80640 gccacctctt ccaccacgag atcttcagga aatggcaggc cactgggttt acatgcagat 80700 ggcatgggag cacacaaggc acggctgtgg ggagttggca cttgctccag aatatggagc 80760 accgagtgaa ggtttcagtt tcctgcactg agagaaacaa gggcattccg aggcttttcc 80820 actttatccc taaagagttt cacaacgctt gtttgccgat ttctacatag atgccacctt 80880 tctgagttgt atgtatttac atgccaaatg tattcattga gcagcgttaa ataatggtgt 80940 tcacccctaa agtgcatata ctggtaaaat taagaatgat cgtaattaag cctcttgcaa 81000 tagtcattag ttcagagaat atttaagaat attaaaggtg ctttgctaat gtcctcgtta 81060 gttttgtttt gacaaaatca gtacttcagt ttcttgtttc tttttttttt gagacggagt 81120 cttactctcg ctctgtcgcc cagactggag actggagtgc agtggcacga tcttggctca 81180 ctgcaacgtc cacctcccag gttcaagcga ttctcctgcc tcagcctccc gagtagctgg 81240 ggttacaggc acacactatg cctggctaat tttttttttt tttttgagac ggagtctcgc 81300 tctgtcaccc aggctggagt gcagtggcgc aatgtcggct cactgcaagc tctgcctcct 81360 gggttcacgc cattctcctg cctcagcctc ccgagtagct gggactacag gcgcccgcca 81420 ccacacccag ctaatttttt tgtattttta gtagagacgg ggtttcacca tgctggccag 81480 gctggtctcg aactcttgac ctcaggtaaa ccacccacct cagcctccca aagttctggg 81540 attacaggcg tgagccacca tgcccagccc agtacttcag tttcttagcg atgaaatcca 81600 cccaatgtca ggcgatgact attattattt tactgattta tactgtttgt tctctattaa 81660 tgtcttattt tccccaaccg attttgaagt tgagtaagga ctatgttccg cgggtatctt 81720 gagtcctctg aggcactgag cttggtgatt tggacgcagg agctgctcat tagtgagctg 81780 atagctggga gcatagcgca tcccacatca cctgacttac cttggtgtcc tcctttgtag 81840 ccttttgtca tacatgatat ggagacactg tgtatggctg agaagacgct ggtggccaag 81900 ctggtggcca atggcatcca gaacaaggag gcggaggtcc gcatctttca ctgctgccag 81960 tgcacgtcag tggagaccgt cacggagctc acggaattcg ccaaggccat cccaggcttc 82020 gcaaacttgg acctgaacga tcaagtgaca ttgctaaaat acggagttta tgaggccata 82080 ttcgccatgc tgtcttctgt gatgaacaaa gacgggatgc tggtagcgta tggaaatggg 82140 tttataactc gtgaattcct aaaaagccta aggaaaccgt tctgtgatat catggaaccc 82200 aagtttgatt ttgccatgaa gttcaatgca ctggaactgg atgacagtga tatctccctt 82260 tttgtggctg ctatcatttg ctgtggaggt gagtggttga tttaatctgc tggtatcatg 82320 tcactgacag gctcctgtct tgaaaaattt gacaatggga aatccagtac cagcctgagc 82380 tgttccagtg gaggggacac tcacatggtg ggaagacgtc tgacccccag tcactgctga 82440 gaattcagtg ggaattataa caatattgta taatattata gtatatattg ttattatcta 82500 taaatacata tttaatatta tgtaaatgta tgacatttta atcataatat tagccaggtg 82560 tgggggtgca cacctttagt cccagctact tactcagtag actgaggcaa aaggatctct 82620 tgagcccagg agttcaggtt gcaatgagtt atgaatgcac cactgcactc tagcctgggc 82680 aacagaacaa gacctatttc tttaaaaaaa aattatatat tttgcacaaa tatatatata 82740 gagaaaaaga ggtcggacat gggcctgtaa tcccagccct ttgggaggct gaggtgggtg 82800 gatcacttga gcccaggagg ttgagaccag cctgggcaac atggcaagac cccgtctcta 82860 caaaaaaaaa aatagaaaaa attagtcaag tatggtggca tgtacctgta gtcccagcta 82920 cttgagaggc tgaggtggga ggatcactta agcccaggag acaaaggttg cagtgagcca 82980 aggtcacgcc accacactcc agcctgggcg acgaagaatg accctgtctc aaaaaaaaaa 83040 aaaaaaaaaa ttatacacac acacacacac acacatttcg tttatattat atctaatatt 83100 ataaacagat ataatttata tattatgata ttcctgtata tattatataa tgatgttgta 83160 ttcatattat agacaatatt gtatgaagtg ctatacagat gtcagtatag ttgctgtcac 83220 agttggttat gttgatgaaa agtatatttc ctaatgcaaa atataatatc agtcagcagc 83280 caagtggcag tgactgcaag gtttgctttg cccgaggaag cagatcccag ggaaggccga 83340 tctggtcctc tctgtggaag ctggctctgc agcctccaca tttttggctc ggtgtcacgt 83400 tcctttaaat agccccatct caggtctagg aaggtcatcc acctactgca aactcggctg 83460 accttaccca gggttggtgg agacagatgg ggtctcccac actgcctgca gccatactgc 83520 gcctggggga ttgactcact gtcagcatgg agctgactca gccctaccag ccgtgcccgt 83580 tactgtgtgg ctgggcacaa gtcagatgaa ggaagtcctt gcgctctggc ataaagtgta 83640 caaagacaaa gcagttatgc ataatttgtc ctttagtatg gtcaggatgt agcattgtgg 83700 gtaaaatgca gttgcagaac tatttatatg tagcatgatc acagttttat aaaggaaatt 83760 ataatcctat atcaatccta tgtatataga aaaatgtcca gtgagatata tgttaaacct 83820 attatggtgg gattaaaatt atgagggggg atttctattt ttcaaaagat tcctcctttt 83880 tttttttttt gagacagagt ctccgtctgt caccctggct ggagtgcagt ggcacgatct 83940 caggtcactg caacttccgc ctcctgggtt caagtgattc gcctgcctca gcctcctgag 84000 tagctgagat tacaggaaca tgccagcaca cctggctaat ttttgtattt ttagtaaaga 84060 tggggtttca ccatattggc caggctggtc tcaaactcct gaccccgggt gacccaccca 84120 cctcggcctc ccaaagtgct gggattacag gcatgagcca ctgcacccgg caataattcc 84180 tctctttaga gacttaatag ttatagcccc agccactctg gaggccgagg caggaggatt 84240 gcttgagcct aggagttcca gtccagccta agcaacagag caagacccca tcactaaaac 84300 aatacaaaaa caagaatttt agaaataaaa acttaataat tacatttaca accaaaaaca 84360 atgaagatgt ttaaatcctc atcactagca accctgttaa gaatcatagt aatgactggg 84420 tctgtaaggg agcaccgcct gctgaacatg gctcagggca gtattttctg gaccaagaat 84480 caggtctcat gctttgagac tgtcccagga tgtctagtgc cagctacccc aggcaggtca 84540 tctggtgtga atgttgactc ttcctgcacc aagtctcaga cctgccccac cctcctcccc 84600 actctgggtc tcctgatctt ggctcactgc aatctccgtc tcccaggttc aagcgattct 84660 cccacctcag cctcccgagt atctgggatt acaggcgtga gccaccgtgc ctggcctaca 84720 aaacctagtt ctaacacaat cactccttaa atatggtgga acacttgaag cttgatatct 84780 agtttggatt caaaagcttc atttcccata ttatgcaaaa ctggtggttg tgatctccag 84840 aatgtactgt tcctcctact agctctaatt tttctccctg acaggtggtc atcaggtaaa 84900 tcacaagtga aaaggccgca ccataaggtg tacttagggc actattgccg cctagtagta 84960 tgaatattta ggaaagagta ctggtcctgt ctgtccctac ttcacctatt gactttggaa 85020 aaacctatgt ctatcttcca gtcaagttga caatatctaa aggcagctca gtttttttct 85080 aagaaaggcc acataaaata ggcatgtttg gttcctgaaa ctgataagca gttcttgggt 85140 gattatcaca ctcaaacctc tctctcttct ttcgagacta gatcgtcctg gccttctaaa 85200 cgtaggacac attgaaaaaa tgcaggaggg tattgtacat gtgctcagac tccacctgca 85260 gagcaaccac ccggacgata tctttctctt cccaaaactt cttcaaaaaa tggcagacct 85320 ccggcagctg gtgacggagc atgcgcagct ggtgcagatc atcaagaaga cggagtcgga 85380 tgctgcgctg cacccgctac tgcaggagat ctacagggac atgtactgag ttccttcaga 85440 tcagccacac cttttccagg agttctgaag ctgacagcac tacaaaggag acgggggagc 85500 agcacgattt tgcacaaata tccaccactt taaccttaga gcttggacag tctgagctgt 85560 aggtaaccgg catattattc catatctttg ttttaaccag tacttctaag agcatagaac 85620 tcaaatgctg ggggtaggtg gctaatctca ggactgggaa gattacggcg aattatgctc 85680 aatggtctga ttttaactca cccgatgtta atcaatgcac attgctttag atcacattcg 85740 tgatttacca tttaattaac tggtaacctc aaaattcgtg gcctgtcttc ccattcaccc 85800 cgcttttgac tattgtgctc ctttataatt ctgaaaacta atcagcactt tttaacaatg 85860 tttataatcc tataagtcta gatgtatcca aaggtgaagt atgtaaaaag cagcaaaata 85920 tttatttcaa agacttcact tctgtttcct gaatctaaag aaagacaaca tgctgctttt 85980 taatcatagg atggagaatt t 86001 <210> SEQ ID NO 5 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 ggcgatctag agagcccgtt a 21 <210> SEQ ID NO 6 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 gccgatggat tgcgaaat 18 <210> SEQ ID NO 7 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 7 aagagttcct gcaagaaatg ggaaacatcc a 31 <210> SEQ ID NO 8 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 9 gaagatggtg atgggatttc 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 10 caagcttccc gttctcagcc 20 <210> SEQ ID NO 11 <211> LENGTH: 2081 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (167)...(1573) <400> SEQUENCE: 11 gtcacagcct aggctttgct ggggacctga gaaacgctgc cgccaagttg aagttcaagg 60 ccctgccttc cctgtgaact gacgtttgtg gctggtcaag ttcgggaaca agacgttgtc 120 atcacagctt agcgctctgt ggcctgcctg gccacatcca tccaac atg gtg gac 175 Met Val Asp 1 aca gag agc ccc atc tgt cct ctc tcc cca ctg gag gca gat gac ctg 223 Thr Glu Ser Pro Ile Cys Pro Leu Ser Pro Leu Glu Ala Asp Asp Leu 5 10 15 gaa agt ccc tta tct gaa gaa ttc tta caa gaa atg gga aac att caa 271 Glu Ser Pro Leu Ser Glu Glu Phe Leu Gln Glu Met Gly Asn Ile Gln 20 25 30 35 gag att tct cag tcc atc ggt gag gag agc tct gga agc ttt ggt ttt 319 Glu Ile Ser Gln Ser Ile Gly Glu Glu Ser Ser Gly Ser Phe Gly Phe 40 45 50 gca gac tac cag tac tta gga agc tgt ccg ggc tcc gag ggc tct gtc 367 Ala Asp Tyr Gln Tyr Leu Gly Ser Cys Pro Gly Ser Glu Gly Ser Val 55 60 65 atc aca gac acc ctc tct cca cgt tcc agc cct tcc tca gtc agc tgc 415 Ile Thr Asp Thr Leu Ser Pro Arg Ser Ser Pro Ser Ser Val Ser Cys 70 75 80 ccc gtg atc ccc gcc agc acg gac gag tcc ccc ggc agt gcc ctg aac 463 Pro Val Ile Pro Ala Ser Thr Asp Glu Ser Pro Gly Ser Ala Leu Asn 85 90 95 atc gag tgt cga ata tgt ggg gac aag gcc tca ggg tac cac tac gga 511 Ile Glu Cys Arg Ile Cys Gly Asp Lys Ala Ser Gly Tyr His Tyr Gly 100 105 110 115 gtt cac gca tgt gaa ggc tgt aag ggc ttc ttt cgg cga act att cgg 559 Val His Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Ile Arg 120 125 130 ctg aag ctg gtg tac gac aag tgt gat cgg agc tgc aag att cag aag 607 Leu Lys Leu Val Tyr Asp Lys Cys Asp Arg Ser Cys Lys Ile Gln Lys 135 140 145 aag aac cgg aac aaa tgc cag tac tgc cgt ttt cac aag tgc ctg tct 655 Lys Asn Arg Asn Lys Cys Gln Tyr Cys Arg Phe His Lys Cys Leu Ser 150 155 160 gtc ggg atg tca cac aat gca att cgc ttt gga aga atg cca aga tct 703 Val Gly Met Ser His Asn Ala Ile Arg Phe Gly Arg Met Pro Arg Ser 165 170 175 gaa aaa gca aaa ctg aaa gca gaa att ctt acc tgt gaa cac gac ctg 751 Glu Lys Ala Lys Leu Lys Ala Glu Ile Leu Thr Cys Glu His Asp Leu 180 185 190 195 aaa gat tcg gaa act gca gac ctc aaa tct ctg ggc aag aga atc cac 799 Lys Asp Ser Glu Thr Ala Asp Leu Lys Ser Leu Gly Lys Arg Ile His 200 205 210 gaa gcc tac ctg aag aac ttc aac atg aac aag gtc aag gcc cgg gtc 847 Glu Ala Tyr Leu Lys Asn Phe Asn Met Asn Lys Val Lys Ala Arg Val 215 220 225 ata ctc gcg gga aag acc agc aac aac ccg cct ttt gtc ata cat gac 895 Ile Leu Ala Gly Lys Thr Ser Asn Asn Pro Pro Phe Val Ile His Asp 230 235 240 atg gag acc ttg tgt atg gcc gag aag acg ctt gtg gcc aag atg gtg 943 Met Glu Thr Leu Cys Met Ala Glu Lys Thr Leu Val Ala Lys Met Val 245 250 255 gcc aac ggc gtc gaa gac aaa gag gca gag gtc cga ttc ttc cac tgc 991 Ala Asn Gly Val Glu Asp Lys Glu Ala Glu Val Arg Phe Phe His Cys 260 265 270 275 tgc cag tgc atg tcc gtg gag acc gtc acg gag ctc aca gaa ttt gcc 1039 Cys Gln Cys Met Ser Val Glu Thr Val Thr Glu Leu Thr Glu Phe Ala 280 285 290 aag gct atc cca ggc ttt gca aac ttg gac ttg aac gac caa gtc acc 1087 Lys Ala Ile Pro Gly Phe Ala Asn Leu Asp Leu Asn Asp Gln Val Thr 295 300 305 ttg cta aag tac ggt gtg tat gaa gcc atc ttc acg atg ctg tcc tcc 1135 Leu Leu Lys Tyr Gly Val Tyr Glu Ala Ile Phe Thr Met Leu Ser Ser 310 315 320 ttg atg aac aaa gac ggg atg ctg atc gcg tac ggc aat ggc ttt atc 1183 Leu Met Asn Lys Asp Gly Met Leu Ile Ala Tyr Gly Asn Gly Phe Ile 325 330 335 aca cgc gag ttc ctt aag aac ctg agg aag ccg ttc tgt gac atc atg 1231 Thr Arg Glu Phe Leu Lys Asn Leu Arg Lys Pro Phe Cys Asp Ile Met 340 345 350 355 gaa ccc aag ttt gac ttc gct atg aag ttc aat gcc tta gaa ctg gat 1279 Glu Pro Lys Phe Asp Phe Ala Met Lys Phe Asn Ala Leu Glu Leu Asp 360 365 370 gac agt gac att tcc ctg ttt gtg gct gct ata att tgc tgt gga gat 1327 Asp Ser Asp Ile Ser Leu Phe Val Ala Ala Ile Ile Cys Cys Gly Asp 375 380 385 cgg cct ggc ctt cta aac ata ggc tac att gag aag ttg cag gag ggg 1375 Arg Pro Gly Leu Leu Asn Ile Gly Tyr Ile Glu Lys Leu Gln Glu Gly 390 395 400 att gtg cac gtg ctt aag ctc cac ctg cag agc aac cat cca gat gac 1423 Ile Val His Val Leu Lys Leu His Leu Gln Ser Asn His Pro Asp Asp 405 410 415 acc ttc ctc ttc cca aag ctc ctt caa aaa atg gtg gac ctt cgg cag 1471 Thr Phe Leu Phe Pro Lys Leu Leu Gln Lys Met Val Asp Leu Arg Gln 420 425 430 435 ctg gtc acg gag cat gcg cag ctc gta cag gtc atc aag aag acc gag 1519 Leu Val Thr Glu His Ala Gln Leu Val Gln Val Ile Lys Lys Thr Glu 440 445 450 tcc gac gca gcg ctg cac cca ctg ttg caa gag atc tac aga gac atg 1567 Ser Asp Ala Ala Leu His Pro Leu Leu Gln Glu Ile Tyr Arg Asp Met 455 460 465 tac tga tctttcctga gatggcaggc cattaccact gttcagggac ctccgaggcc 1623 Tyr * tgcggcccca tacaggagag cagggatttg cacagagggc ctccctccta cgcttgggga 1683 tgaagagggc tgagcgtagg taatgcgggc tctccccaca tcctttctga atgggcactt 1743 ctaagactac ctgctaccga aatgggggtg atcggaggct aataggattc agacagtgac 1803 agacaacggc agtccccagt ctggtcttaa ccggcccaat gttaatcaat gcacagcact 1863 ctacgttgcg tttataattc gccattaatt aacgggtaac ctcgaagtct gagcggtctg 1923 ttcccttcct gccacccttc tggctatgtg cactctctta aatccctgaa aactaatctg 1983 cactttttaa cctttgaaaa cctacaagtc aaggtgtggc ccaaggttag ccatttaaat 2043 gtggcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2081 <210> SEQ ID NO 12 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 12 aacgggtaac ctcgaagtct ga 22 <210> SEQ ID NO 13 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 13 agggatttaa gagagtgcac atagc 25 <210> SEQ ID NO 14 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 14 cggtctgttc ccttcctgcc acc 23 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 15 ggcaaattca acggcacagt 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 16 gggtctcgct cctggaagat 20 <210> SEQ ID NO 17 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 17 aaggccgaga atgggaagct tgtcatc 27 <210> SEQ ID NO 18 <211> LENGTH: 1850 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (213)...(1619) <400> SEQUENCE: 18 ggcccaggct gaagctcagg gccctgtctg ctctgtggac tcaacagttt gtggcaagac 60 aagctcagaa ctgagaagct gtcaccacag ttctggaggc tgggaagttc aagatcaaag 120 tgccagcaga ttcagtgtca tgtgaggacg tgcttcctgc ttcatagata agagcttgga 180 gctcggcgca caaccagcac catctggtcg cg atg gtg gac acg gaa agc cca 233 Met Val Asp Thr Glu Ser Pro 1 5 ctc tgc ccc ctc tcc cca ctc gag gcc ggc gat cta gag agc ccg tta 281 Leu Cys Pro Leu Ser Pro Leu Glu Ala Gly Asp Leu Glu Ser Pro Leu 10 15 20 tct gaa gag ttc ctg caa gaa atg gga aac atc caa gag att tcg caa 329 Ser Glu Glu Phe Leu Gln Glu Met Gly Asn Ile Gln Glu Ile Ser Gln 25 30 35 tcc atc ggc gag gat agt tct gga agc ttt ggc ttt acg gaa tac cag 377 Ser Ile Gly Glu Asp Ser Ser Gly Ser Phe Gly Phe Thr Glu Tyr Gln 40 45 50 55 tat tta gga agc tgt cct ggc tca gat ggc tcg gtc atc acg gac acg 425 Tyr Leu Gly Ser Cys Pro Gly Ser Asp Gly Ser Val Ile Thr Asp Thr 60 65 70 ctt tca cca gct tcg agc ccc tcc tcg gtg act tat cct gtg gtc ccc 473 Leu Ser Pro Ala Ser Ser Pro Ser Ser Val Thr Tyr Pro Val Val Pro 75 80 85 ggc agc gtg gac gag tct ccc agt gga gca ttg aac atc gaa tgt aga 521 Gly Ser Val Asp Glu Ser Pro Ser Gly Ala Leu Asn Ile Glu Cys Arg 90 95 100 atc tgc ggg gac aag gcc tca ggc tat cat tac gga gtc cac gcg tgt 569 Ile Cys Gly Asp Lys Ala Ser Gly Tyr His Tyr Gly Val His Ala Cys 105 110 115 gaa ggc tgc aag ggc ttc ttt cgg cga acg att cga ctc aag ctg gtg 617 Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Val 120 125 130 135 tat gac aag tgc gac cgc agc tgc aag atc cag aaa aag aac aga aac 665 Tyr Asp Lys Cys Asp Arg Ser Cys Lys Ile Gln Lys Lys Asn Arg Asn 140 145 150 aaa tgc cag tat tgt cga ttt cac aag tgc ctt tct gtc ggg atg tca 713 Lys Cys Gln Tyr Cys Arg Phe His Lys Cys Leu Ser Val Gly Met Ser 155 160 165 cac aac gcg att cgt ttt gga cga atg cca aga tct gag aaa gca aaa 761 His Asn Ala Ile Arg Phe Gly Arg Met Pro Arg Ser Glu Lys Ala Lys 170 175 180 ctg aaa gca gaa att ctt acc tgt gaa cat gac ata gaa gat tct gaa 809 Leu Lys Ala Glu Ile Leu Thr Cys Glu His Asp Ile Glu Asp Ser Glu 185 190 195 act gca gat ctc aaa tct ctg gcc aag aga atc tac gag gcc tac ttg 857 Thr Ala Asp Leu Lys Ser Leu Ala Lys Arg Ile Tyr Glu Ala Tyr Leu 200 205 210 215 aag aac ttc aac atg aac aag gtc aaa gcc cgg gtc atc ctc tca gga 905 Lys Asn Phe Asn Met Asn Lys Val Lys Ala Arg Val Ile Leu Ser Gly 220 225 230 aag gcc agt aac aat cca cct ttt gtc ata cat gat atg gag aca ctg 953 Lys Ala Ser Asn Asn Pro Pro Phe Val Ile His Asp Met Glu Thr Leu 235 240 245 tgt atg gct gag aag acg ctg gtg gcc aag ctg gtg gcc aat ggc atc 1001 Cys Met Ala Glu Lys Thr Leu Val Ala Lys Leu Val Ala Asn Gly Ile 250 255 260 cag aac aag gag gcg gag gtc cgc atc ttt cac tgc tgc cag tgc acg 1049 Gln Asn Lys Glu Ala Glu Val Arg Ile Phe His Cys Cys Gln Cys Thr 265 270 275 tca gtg gag acc gtc acg gag ctc acg gaa ttc gcc aag gcc atc cca 1097 Ser Val Glu Thr Val Thr Glu Leu Thr Glu Phe Ala Lys Ala Ile Pro 280 285 290 295 ggc ttc gca aac ttg gac ctg aac gat caa gtg aca ttg cta aaa tac 1145 Gly Phe Ala Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr 300 305 310 gga gtt tat gag gcc ata ttc gcc atg ctg tct tct gtg atg aac aaa 1193 Gly Val Tyr Glu Ala Ile Phe Ala Met Leu Ser Ser Val Met Asn Lys 315 320 325 gac ggg atg ctg gta gcg tat gga aat ggg ttt ata act cgt gaa ttc 1241 Asp Gly Met Leu Val Ala Tyr Gly Asn Gly Phe Ile Thr Arg Glu Phe 330 335 340 cta aaa agc cta agg aaa ccg ttc tgt gat atc atg gaa ccc aag ttt 1289 Leu Lys Ser Leu Arg Lys Pro Phe Cys Asp Ile Met Glu Pro Lys Phe 345 350 355 gat ttt gcc atg aag ttc aat gca ctg gaa ctg gat gac agt gat atc 1337 Asp Phe Ala Met Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Ile 360 365 370 375 tcc ctt ttt gtg gct gct atc att tgc tgt gga gat cgt cct ggc ctt 1385 Ser Leu Phe Val Ala Ala Ile Ile Cys Cys Gly Asp Arg Pro Gly Leu 380 385 390 cta aac gta gga cac att gaa aaa atg cag gag ggt att gta cat gtg 1433 Leu Asn Val Gly His Ile Glu Lys Met Gln Glu Gly Ile Val His Val 395 400 405 ctc aga ctc cac ctg cag agc aac cac ccg gac gat atc ttt ctc ttc 1481 Leu Arg Leu His Leu Gln Ser Asn His Pro Asp Asp Ile Phe Leu Phe 410 415 420 cca aaa ctt ctt caa aaa atg gca gac ctc cgg cag ctg gtg acg gag 1529 Pro Lys Leu Leu Gln Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu 425 430 435 cat gcg cag ctg gtg cag atc atc aag aag acg gag tcg gat gct gcg 1577 His Ala Gln Leu Val Gln Ile Ile Lys Lys Thr Glu Ser Asp Ala Ala 440 445 450 455 ctg cac ccg cta ctg cag gag atc tac agg gac atg tac tga gttccttcag 1629 Leu His Pro Leu Leu Gln Glu Ile Tyr Arg Asp Met Tyr 460 465 atcagccaca ccttttccag gagttctgaa gctgacagca ctacaaagga gacgggggag 1689 cagcacgatt ttgcacaaat atccaccact ttaaccttag agcttggaca gtctgagctg 1749 taggtaaccg gcatattatt ccatatcttt gttttaacca gtacttctaa gagcatagaa 1809 ctcaaatgct gggggaggtg gctaatctca ggactgggaa g 1850 <210> SEQ ID NO 19 <211> LENGTH: 417 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <400> SEQUENCE: 19 ggcgggcggc gggggcggag gcggccgcta gcgccctgcc cgggccgcct ccttcggcgt 60 tcgccccacg gaccggcagg cggcggaccg cggcccaggc tgaagctcag ggccctgtct 120 gctctgtgga ctcaacagtt tgtggcaaga caagctcaga actgagaagc tgtcaccaca 180 gtagcttgga gctcggcggc acaaccagca ccatctggtc gcgatggtgg acacggaaag 240 cccactctgc cccctctccc cactcgaggc cggcgatcta gagagcccgt tatctgaaga 300 gttcctgcaa gaaatgggaa acatccaaga gatttcgcaa tccatcggcg aggatagttc 360 tggaagcttt ggctttacgg aataccagta tttaggaagc tgtcctggct cagatgg 417 <210> SEQ ID NO 20 <211> LENGTH: 1580 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (220)...(996) <400> SEQUENCE: 20 ggcacgaggg aagttcaaga tcaaagtgcc agcagattca gtgtcatgtg aggacgtgct 60 tcctgcttca tagataagag tgccacaccc ccctgtccca gtgcactggg gctgtggatc 120 ccttcaaagc tgagattgcc tgtctgtggt ctccagcgtt aagcacagtc attagctcag 180 cttggagctc ggcggcacaa ccagcaccat ctggtcgcg atg gtg gac acg gaa 234 Met Val Asp Thr Glu 1 5 agc cca ctc tgc ccc ctc tcc cca ctc gag gcc ggc gat cta gag agc 282 Ser Pro Leu Cys Pro Leu Ser Pro Leu Glu Ala Gly Asp Leu Glu Ser 10 15 20 ccg tta tct gaa gag ttc ctg caa gaa atg gga aac atc caa gag att 330 Pro Leu Ser Glu Glu Phe Leu Gln Glu Met Gly Asn Ile Gln Glu Ile 25 30 35 tcg caa tcc atc ggc gag gat agt tct gga agc ttt ggc ttt acg gaa 378 Ser Gln Ser Ile Gly Glu Asp Ser Ser Gly Ser Phe Gly Phe Thr Glu 40 45 50 tac cag tat tta gga agc tgt cct ggc tca gat ggc tcg gtc atc acg 426 Tyr Gln Tyr Leu Gly Ser Cys Pro Gly Ser Asp Gly Ser Val Ile Thr 55 60 65 gac acg ctt tca cca gct tcg agc ccc tcc tcg gtg act tat cct gtg 474 Asp Thr Leu Ser Pro Ala Ser Ser Pro Ser Ser Val Thr Tyr Pro Val 70 75 80 85 gtc ccc ggc agc gtg gac gag tct ccc agt gga gca ttg aac atc gaa 522 Val Pro Gly Ser Val Asp Glu Ser Pro Ser Gly Ala Leu Asn Ile Glu 90 95 100 tgt aga atc tgc ggg gac aag gcc tca ggc tat cat tac gga gtc cac 570 Cys Arg Ile Cys Gly Asp Lys Ala Ser Gly Tyr His Tyr Gly Val His 105 110 115 gcg tgt gaa ggc tgc aag ggc ttc ttt cgg cga acg att cga ctc aag 618 Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Ile Arg Leu Lys 120 125 130 ctg gtg tat gac aag tgc gac cgc agc tgc aag atc cag aaa aag aac 666 Leu Val Tyr Asp Lys Cys Asp Arg Ser Cys Lys Ile Gln Lys Lys Asn 135 140 145 aga aac aaa tgc cag tat tgt cga ttt cac aag tgc ctt tct gtc ggg 714 Arg Asn Lys Cys Gln Tyr Cys Arg Phe His Lys Cys Leu Ser Val Gly 150 155 160 165 atg tca cac aac gcg att cgt ttt gga cga atg cca aga tct gag aaa 762 Met Ser His Asn Ala Ile Arg Phe Gly Arg Met Pro Arg Ser Glu Lys 170 175 180 gca aaa ctg aaa gca gaa att ctt acc tgt gaa cat gac ata gaa gat 810 Ala Lys Leu Lys Ala Glu Ile Leu Thr Cys Glu His Asp Ile Glu Asp 185 190 195 tct gaa act gca gat ctc aaa tct ctg gcc aag aga atc tac gag gcc 858 Ser Glu Thr Ala Asp Leu Lys Ser Leu Ala Lys Arg Ile Tyr Glu Ala 200 205 210 tac ttg aag aac ttc aac atg aac aag gtc aaa gcc cgg gtc atc ctc 906 Tyr Leu Lys Asn Phe Asn Met Asn Lys Val Lys Ala Arg Val Ile Leu 215 220 225 tca gga aag gcc agt aac aat cca gta ggt gtt tgc ggc tgt tct ggg 954 Ser Gly Lys Ala Ser Asn Asn Pro Val Gly Val Cys Gly Cys Ser Gly 230 235 240 245 ttc tct tgg caa cat gga acc agt gtc gta gag gac gat taa 996 Phe Ser Trp Gln His Gly Thr Ser Val Val Glu Asp Asp * 250 255 ggacacatgt gttgaatgtt gagaaaatta tatttatccc acagttaagc aaaggacagc 1056 gaagatggaa acagttcatt ctgagactct gagctgtagc ttaacaacaa ctcctttctt 1116 cttgcttgga gccacctcaa agctcttagc aactaagtta ttatactggc tatgtaatta 1176 atacacttaa aaaaaacctt aatagcttac caagtactaa gatgatttct taggagcatt 1236 ttttcttaaa tagagatagg ttcttgctct gttgcccagg ctggaatgca gtggtgcaat 1296 catagttcac tgcagccttg aactcctggg ctcaagcaat cctcctgcct cagcctccca 1356 aggagctggg actacaggtg tgcaccacca cacctggcta tgtttgatgt tgttgttgtt 1416 ttgttttgtt tttgtttttt ggtagagatg agatgtttcc caggctggtc tcaaactcct 1476 ggcctcaagt gatcttccca cctcggcctc ccaaagcact ggcattacag gtgtgagtca 1536 tggcacccag cattaactgg atttaaaaaa aaaaaaaaaa aaaa 1580 <210> SEQ ID NO 21 <211> LENGTH: 232 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: 5′UTR <222> LOCATION: (1)...(232) <400> SEQUENCE: 21 gttgtcccct cggagggagg gcccacgggc ggggacatcg ggacttgccc tttcctcggc 60 gcagcggagc tggggcgtcg ccgactcaga aggtgctttc cgagacctcc agggatctcc 120 gaggcgagga aacccgggcc ccggacagac cgaccctggg ctgaagctca gggccctgtc 180 tgctctgtgg actcaacagt ttgtggcaag acaagctcag aactgagaag ct 232 <210> SEQ ID NO 22 <211> LENGTH: 547 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <400> SEQUENCE: 22 tttgagtcct ctgaggcact gagcttggtg atttggacgc aggagctgct cattagtgag 60 ctgatagctg ggagcatagc gcatcccaca tcacctgact taccttggtg tcctcctttg 120 tagccttttg tcatacatga tatggagaca ctgtgtatgg ctgagaagac gctggtggcc 180 aagctggtgg ccaatggcat ccagaacaag gaggcggagg tccgcatctt tcactgctgc 240 cagtgcacgt cagtggagac cgtcacggag ctcacggaat tcgccaaggc catcccaggc 300 ttcgcaaact tggacctgaa cgatcaagtg acattgctaa aatacggagt ttatgaggcc 360 atattcgcca tgctgtcttc tgtgatgaac aaagacggga tgctggtagc gtatggaaat 420 gggtttataa ctcgtgaatt cctaaaaagc ctaaggaaac cgttctgtga tatcatggaa 480 cccaagtttg attttgccat gaagttcaat gcactggaac tggatgacag tgatatctcc 540 ctttttg 547 <210> SEQ ID NO 23 <211> LENGTH: 731 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <400> SEQUENCE: 23 ctcaaatctc tggccaagag aatctacgag gcctacttga agaacttcaa catgaacaag 60 gtcaaagccc gggtcatcct ctcaggaaag gccagtaaca atccagtagg tgtttgcggc 120 tgttctgggt tctcttggca acatggaacc agtgtcgtag aggacgatta aggacacatg 180 tgttgaatgt tgagaaaatt atatttatcc cacagttaag caaaggacag cgaagatgga 240 aacagttcat tctgagactc tgagctgtag cttaacaaca actcctttct tcttgcttgg 300 agccacctca aagctcttag caactaagtt attatactgg ctatgtaatt aatacactta 360 aaaaaaacct taatagctta ccaagtacta agatgatttc ttaggagcat tttttcttaa 420 atagagatag gttcttgctc tgttgcccag gctggaatgc agtggtgcaa tcatagttca 480 ctgcagcctt gaactcctgg gctcaagcaa tcctcctgcc tcggctccca aggagctggg 540 actacaggtg tgcaccacca cacctggcta tgtttgatgt tgttgtgttg ttgttggttt 600 ggtagagatg agatgtttcc cagctggtct caaactcctg gctcaagtga tcttcccacc 660 tcggcttcca agcactggca ttacaggtgt gagtcatggc accagcatta ctggattaaa 720 aaaaaaaaaa a 731 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 cagggccctg agcttcagcc 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 cacaaactgt tgagtccaca 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 gacagcttct cagttctgag 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 gcgccgagct ccaagctctt 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 gtgtccacca tcgcgaccag 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 ttgcaggaac tcttcagata 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 tatcctcgcc gatggattgc 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 aagcttccag aactatcctc 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 ttcctaaata ctggtattcc 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 ccgagccatc tgagccagga 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 ccgtgatgac cgagccatct 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 aaagcgtgtc cgtgatgacc 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 36 ttcaatgctc cactgggaga 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 37 cattcgatgt tcaatgctcc 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 38 cgcagattct acattcgatg 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 39 cgtggactcc gtaatgatag 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 40 cacttgtgaa atcgacaata 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 41 agaaaggcac ttgtgaaatc 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 42 ttgtgtgaca tcccgacaga 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 43 ttggcattcg tccaaaacga 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 tttctcagat cttggcattc 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 cagttttgct ttctcagatc 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 aagaatttct gctttcagtt 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 caggtaagaa tttctgcttt 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 gttcacaggt aagaatttct 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 ctcttggcca gagatttgag 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 tcaagtaggc ctcgtagatt 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 ccttgttcat gttgaagttc 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 tgacaaaagg tggattgtta 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 ccattggcca ccagcttggc 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 ggacctccgc ctccttgttc 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 atggccttgg cgaattccgt 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 gttcaggtcc aagtttgcga 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 tccgtatttt agcaatgtca 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 gcctcataaa ctccgtattt 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 cacagaagac agcatggcga 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 catcccgtct ttgttcatca 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 catttccata cgctaccagc 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 agaacggttt ccttaggctt 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 gcagccacaa aaagggagat 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 gcaaatgata gcagccacaa 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 tttagaaggc caggacgatc 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 caataccctc ctgcattttt 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 ctgagcacat gtacaatacc 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 gcaggtggag tctgagcaca 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 gctctgcagg tggagtctga 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 ctccgtcacc agctgccgga 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 ttgatgatct gcaccagctg 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 72 tgaaggaact cagtacatgt 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 aactcctgga aaaggtgtgg 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 ggtggatatt tgtgcaaaat 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 ctgtccaagc tctaaggtta 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 taatatgccg gttacctaca 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 tcccccagca tttgagttct 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 78 gcgcacccac ccagggtcgg 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 79 ttctatttac ctgtggtgac 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 80 aattctgtgc ccaagtttcc 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 81 taaacgtgta tgtacctctt 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 82 gatgatgctt acagtgttca 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 83 caaagaactt gtgaccattt 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 84 gtgtggcact ggcacgggaa 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 85 gagtacgcac ctgagctaat 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 86 ctccaagcta ctgggaggaa 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 87 gaaagaagcc ctgtgagggt 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 88 atgtcactgt cttttcactg 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 89 agcttcagcc tgggccgcgg 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 90 ctccaagcta ctgtggtgac 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 91 tcttgaactt ccctcgtgcc 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 92 gggtgtggca ctcttatcta 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 93 acgctggaga ccacagacag 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 94 gagctccaag ctgagctaat 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 95 cgacactggt tccatgttgc 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 96 gaatgaactg tttccatctt 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 97 gcttcagccc agggtcggtc 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 98 atgtgggatg cgctatgctc 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 99 tggtaagcta ttaaggtttt 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 100 ttagtacttg gtaagctatt 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 101 tgactcacac ctgtaatgcc 20 <210> SEQ ID NO 102 <211> LENGTH: 4325 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1328-1427, 1533-1632, 1900-1999, 2180-2279, 2440-2539, 2763-2862, 3331-3430 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 102 gcggccgcgc ctccctgcga ccgtcctcga tgcccttcag cctctgcctt cccccgcccg 60 ccacacccac cctggcacct tggccacctg ttgccgcgtg ccccagctcg ctcttccttt 120 ctccccattt ctcatcctgg gactctgaag atcagatttc gcctgtccgt ccacctcccc 180 acgaactccc gggactcggg gaacaagctg tgcgatctta gaccagctca acgagggcac 240 ccggaggaaa gctcaaccca gacccgcagc cttgaacttc agtcctggcc ggtgcgcggg 300 gctgggagca ggagggagtg cgcgccaagg cgcacccttc ccaccgactg ttctcccccg 360 tcgggtgacc ttgggcagtc ccttcaccta acccgcctca gtttaccaac ggatgcccgg 420 gccccgaggc actaaatggg catcgaggag agctgccccg ggcctcttag gccccgccct 480 cccccgcagc agccaatcag acactgccgt ccagggggtg tgtctcgccc tgagccgggg 540 cccgggccta gggggcggag tttccggggc ggtcacctcg ccgcgggacc ccgcagggga 600 cgtccgaggg gcggcgcgtg tcgtgggggc gcggctggca cgggcgcgcg taggcggtgc 660 cgggccgggg ccccggacgc tacggtccca cgacaggggt gacgggggcg gaggcagccg 720 cttacgcccc tcctggcgcc tcctcctggg cgcgcttggc cctgcggacc cgcaggcgga 780 gtgcagcctc aggtgcccag gggctggagg gcacgcgcga gggcggggag ccaggcgtcc 840 cctgtcccgg gacagtgagg tgggtggaca gggaggggag gggctcggtg gcgcatgcgc 900 gcggactagg ggcgcgggtc tggagaccca cagccactgg agagggcaca cgctaggaag 960 ggcacacgcg tgcgagtttt cagggcccgc ggaactgtcc gccacttcga gtcccctgga 1020 gcgccgtgcg ccggctccga acattggtgt tcgcagctgt tttgggggct ggagggttcg 1080 tggagtcctg gaactggagc gacgctgggt cctctggttg tcccctggag gggagggcac 1140 acgggcgggg acatcgggcg ctcccttctc acggcgtggt gcatttgggc gtatctcacc 1200 gggaggcgtt tcctgagacc ctcggggaac ttagaggaga ggtaactgga ggctccctga 1260 cagactgatt ctggggtcac agcctaggct ttgctgggga cctgagaaac gctgccggtg 1320 ggtttgannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnncgt tctacagcca 1440 agttgaagtt caaggccctg ccttccctgt gaactgacgt ttgtggctgg tcaagttcgg 1500 gaacaagacg ttgtcatcac aggtaaagag gannnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nncctcccac agcttagcgc tctgtggcct gcctggccac atccatccaa 1680 catggtggac acagagagcc ccatctgtcc tctctcccca ctggaggcag atgacctgga 1740 aagtccctta tctgaagaat tcttacaaga aatgggaaac attcaagaga tttctcagtc 1800 catcggtgag gagagctctg gaagctttgg ttttgcagac taccagtact taggaagctg 1860 tccgggctcc gagggctctg tcatcacagg taagtgctgn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnc catctgtaga caccctctct ccagcttcca gcccttcctc 2040 agtcagctgc cccgtgatcc ccgccagcac ggacgagtcc cccggcagtg ccctgaacat 2100 cgagtgtcga atatgtgggg acaaggcctc agggtaccac tacggagttc acgcatgtga 2160 aggctgtaag taaggaggcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnng 2280 acgtcacagg ggcttctttc ggcgaactat tcggctgaag ctggtgtacg acaagtgtga 2340 tcggagctgc aagattcaga agaagaaccg gaacaaatgc cagtactgcc gttttcacaa 2400 gtgcctgtct gtcgggatgt cacacaatgg taggttaggn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2520 nnnnnnnnnn nnnnnnnnnc caactccagc aattcgcttt ggaagaatgc caagatctga 2580 aaaagcaaaa ctgaaagcag aaattcttac ctgtgaacac gacctgaaag attcggaaac 2640 tgcagacctc aaatctctgg gcaagagaat ccacgaagcc tacctgaaga acttcaacat 2700 gaacaaggtc aaggcccggg tcatactcgc gggaaagacc agcaacaacc cggtaggtgc 2760 ttnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntcccttgt agccttttgt 2880 catacatgac atggagacct tgtgtatggc cgagaagacg cttgtggcca agatggtggc 2940 caacggcgtc gaagacaaag aggcagaggt ccgattcttc cactgctgcc agtgcatgtc 3000 cgtggagacc gtcacggagc tcacagaatt tgccaaggct atcccaggct ttgcaaactt 3060 ggacttgaac gaccaagtca ccttgctaaa gtacggtgtg tatgaagcca tcttcacgat 3120 gctgtcctcc ttgatgaaca aagacgggat gctgatcgcg tacggcaatg gctttatcac 3180 acgcgagttc cttaagaacc tgaggaagcc gttctgtgac atcatggaac ccaagtttga 3240 cttcgctatg aagttcaatg ccttagaact ggatgacagt gacatttccc tgtttgtggc 3300 tgctataatt tgctgtggag gtgagtggtc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3420 nnnnnnnnnn ttgagcgtag atcggcctgg ccttctaaac ataggctaca ttgagaagtt 3480 gcaggagggg attgtgcacg tgcttaagct ccacctgcag agcaaccatc cagatgacac 3540 cttcctcttc ccaaagctcc ttcaaaaaat ggtggacctt cggcagctgg tcacggagca 3600 tgcgcagctc gtacaggtca tcaagaagac cgagtccgac gcagcgctgc acccactgtt 3660 gcaagagatc tacagagaca tgtactgatc tttcctgaga tggcaggccg ttgccactgt 3720 tcagggacct ccgaggcctg cggccccata caggagagca gggatttgca cagagggcct 3780 ccctcctacg cttggggatg aagagggctg agcgtaggta atgcgggctc tccccacatc 3840 ctttctgaat gggcacttct aagactacct gctaccgaaa tgggggtgat cggaggctaa 3900 taggattcag acagtgacag acaatgggag ccccagtctg gtcttaaccg gcccaatgtt 3960 aatcaatgca cagcactcta cgttgcgttt ataattcgcc attaattaac gggtaacctc 4020 gaagtctgag cggtctgttc ccttcctgcc acccttctgg atatgtgcac tctcttaaat 4080 ccctgaaaac taatctgcac tttttaacct ttgaaaacct acaagtcaag gtgtggccca 4140 aggttagcca tttaaatgtg gcaaaaaaaa aaaaatatgt ttattgggaa gacttcactt 4200 gagtttcctg gctctaagaa agagagctgg cttctgagaa cattcgagaa tagtttgata 4260 agctatccca tcactctctc tgtgggctca ctgttctgga gggtgtaact gactcatgag 4320 ggtgg 4325 <210> SEQ ID NO 103 <211> LENGTH: 815 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 475, 735 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 103 gccatcttca cgatgctgtc ctccttgatg aacaaagacg ggatgctgat cgcgtacggc 60 aatggcttta tcacacgcga gttccttaag aacctgagga agccgttctg tgacatcatg 120 gaacccaagt ttgacttcgc tatgaagttc aatgccttag aactggatga cagtgacatt 180 tccctgtttg tggctgctat aatttgctgt ggagctcctt caaaaaatgg tggaccttcg 240 gcagctggtc acggagcatg cgcagctcgt acaggtcatc aagaagaccg agtccgacgc 300 agcgctgcac ccactgttgc aagagatcta cagagacatg tactgatctt tcctgagatg 360 gcaggccgtt gccactgttc agggacctcc gaggcctgcg gccccataca ggagagcagg 420 gatttgcaca gagggcctcc ctcctacgct tggggatgaa gagggctgag cgtangtaat 480 gcgggctctc cccacatcct ttctgaatgg cacttctaga ctacctgcta ccgaaatggg 540 gtgatcggag gctaatagga ttcagacagt gacagacaac ggcagtcccc agtctggtct 600 taacccgccc caatgtaatc aatgcacagc actcttacgt tgcgttataa ttcgccatta 660 attaacgggt accctcaagt ctgagcgggc tggtcccttt cctgcaccct tctggctatg 720 tgcactctct taaantcctg aaaactaatc tggacttttt aacctttgaa aactacaagt 780 caaggtgtgg cccaagttag ccatttaaat gtggc 815 <210> SEQ ID NO 104 <211> LENGTH: 671 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 596, 659 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 104 gggagaccca cagccactgg agagggcaca cgctaggaag ggcacacgcg tgcgagtttt 60 cagggcccgc ggaactgtcc gccacttcga gtcccctgga gcgccgtgcg ccggctccga 120 acattggtgt tcgcagctgt tttgggggct ggagggttcg tggagtcctg gaactggagc 180 gacgctgggt cctctggttg tcccctggag gggagggcac acgggcgggg acatcggggc 240 gctcccttct cacggcgtgg tgcatttggg cgtatctcac cgggaggcgt ttcctgagac 300 cctcggggaa cttagaggag agccaagttg aagttcaagg ccctgccttc cctgtgaact 360 gacgtttgtg gctggtcaag ttcgggaaca agacgttgtc atcacagctt agcgctctgt 420 ggcctgcctg gccacatcca tccaacatgg tggacacaga gagccccatc tgtcctctct 480 ccccactgga ggcagatgac ctggaaagtc ccttatctga agaattctta cacgaaatgg 540 gaaacattca agagatttct cagtccatcg gtgaggagag ctctgcaagc tttggntttg 600 cagactacca gtacttagga agctgtccgg gcttcgaggg ctctgtcatc acagacacnc 660 tctctcagct t 671 <210> SEQ ID NO 105 <211> LENGTH: 676 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <400> SEQUENCE: 105 gggaggcagc cgcttacgcc cctcctggcg cctcctcctg ggcgcgcttg gccctgcgga 60 cccgcaggcg gagtgcagcc tcagccaagt tgaagttcaa ggccctgcct tccctgtgaa 120 ctgacgtttg tggctggtca agttcgggaa caagacgttg tcatcacagc ttagcgctct 180 gtggcctgcc tggccacatc catccaacat ggtggacaca gagagcccca tctgtcctct 240 ctccccactg gaggcagatg acctggaaag tcccttatct gaagaattct tacaagaaat 300 gggaaacatt caagagattt ctcagtccat cggtgaggag agctctggaa gctttggttt 360 tgcagactac cagtacttag gaagctgtcc gggctccgag ggctctgtca tcacagacac 420 cctctctcca gcttccagcc cttcctcagt cagctgcccc gtgatccccg ccagcacgga 480 cgagtccccc ggcagtgccc tgaacatcga gtgtcgaata tgtggggaca aggcctcagg 540 gtaccactac ggagttcacg catgtgaagg ctgtaagggc ttctttcggc gaactattcg 600 gctgaagctg gtgtacgaca agtgtgatcc gagctgctag attcacaaga agaacccgaa 660 ccatgccaga ctgcct 676 <210> SEQ ID NO 106 <211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE: <400> SEQUENCE: 106 gttgctcatc ctgggactct aaagatcaga ttccgcctgt ccgtccacct ccccacgaac 60 tcccgggact cggggaacaa gctgtgcgat ctagaccagc tcaacgaggg cacccggagg 120 aaagctcaac ccagacccgc agccttgaac ttcagtcctg gccgccaagt tgaagttcaa 180 ggccctgcct tccctgtgaa ctgacgtttg tggctggtca agttcgggaa caagacgttg 240 tcatcacagc ttagcgctct gtggcctgcc tggccacatc catccaacat ggtggacaca 300 gagagcccca tctgtcctct ctccccactg gaggcagatg acctggaaag tccttatctg 360 <210> SEQ ID NO 107 <211> LENGTH: 1897 <212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 107 gctaggaagg gcacacgcgt gcgagttttc agggcccgcg gaactgtccg ccacttcgag 60 tcccctggag cgccgtgcgc cggctccgaa cattggtgtt cgcagctgtt ttgggggctg 120 gagggttcgt ggagtcctgg aactggagcg acgctgggtc ctctggttgt cccctggagg 180 ggagggcaca cgggcgggga catcgggcgc tcccttctca cggcgtggtg catttgggcg 240 tatctcaccg ggaggcgttt cctgagaccc tcggggaact tagaggagag gtaactggag 300 gctccctgac agactgattc tggggtcaca gcctaggctt tgctggggac ctgagaaacg 360 ctgccgccaa gttgaagttc aaggccctgc cttccctgtg aactgacgtt tgtggctggt 420 caagttcggg aacaagacgt tgtcatcaca gcttagcgct ctgtggcctg cctggccaca 480 tccatccaac atggtggaca cagagagccc catctgtcct ctctccccac tggaggcaga 540 tgacctggaa agtcccttat ctgaagaatt cttacaagaa atgggaaaca ttcaagagat 600 ttctcagtcc atcggtgagg agagctctgg aagctttggt tttgcagact accagtactt 660 aggaagctgt ccgggctccg agggctctgt catcacagac accctctctc cagcttccag 720 cccttcctca gtcagctgcc ccgtgatccc cgccagcacg gacgagtccc ccggcagtgc 780 cctgaacatc gagtgtcgaa tatgtgggga caaggcctca gggtaccact acggagttca 840 cgcatgtgaa ggctgtaagg gcttctttcg gcgaactatt cggctgaagc tggtgtacga 900 caagtgtgat cggagctgca agattcagaa gaagaaccgg aacaaatgcc agtactgccg 960 ttttcacaag tgcctgtctg tcgggatgtc acacaatgca attcgctttg gaagaatgcc 1020 aagatctgaa aaagcaaaac tgaaagcaga aattcttacc tgtgaacacg acctgaaaga 1080 ttcggaaact gcagacctca aatctctggg caagagaatc cacgaagcct acctgaagaa 1140 cttcaacatg aacaaggtca aggcccgggt catactcgcg ggaaagacca gcaacaaccc 1200 gccttttgtc atacatgaca tggagacctt gtgtatggcc gagaagacgc ttgtggccaa 1260 gatggtggcc aacggcgtcg aagacaaaga ggcagaggtc cgattcttcc actgctgcca 1320 gtgcatgtcc gtggagaccg tcacggagct cacagaattt gccaaggcta tcccaggctt 1380 tgcaaacttg gacttgaacg accaagtcac cttgctaaag tacggtgtgt atgaagccat 1440 cttcacgatg ctgtcctcct tgatgaacaa agacgggatg ctgatcgcgt acggcaatgg 1500 ctttatcaca cgcgagttcc ttaagaacct gaggaagccg ttctgtgaca tcatggaacc 1560 caagtttgac ttcgctatga agttcaatgc cttagaactg gatgacagtg acatttccct 1620 gtttgtggct gctataattt gctgtggaga tcggcctggc cttctaaaca taggctacat 1680 tgagaagttg caggagggga ttgtgcacgt gcttaagctc cacctgcaga gcaaccatcc 1740 agatgacacc ttcctcttcc caaagctcct tcaaaaaatg gtggaccttc ggcagctggt 1800 cacggagcat gcgcagctcg tacaggtcat caagaagacc gagtccgacg cagcgctgca 1860 cccactgttg caagagatct acagagacat gtactga 1897 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 108 aggcagggcc ttgaacttca 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 109 cagttcacag ggaaggcagg 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 110 gtgtccacca tgttggatgg 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 111 ggactttcca ggtcatctgc 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 112 ttcagataag ggactttcca 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 113 gtaagaattc ttcagataag 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 114 gagaaatctc ttgaatgttt 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 115 tctgtgatga cagagccctc 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 116 gagagggtgt ctgtgatgac 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 117 cacatattcg acactcgatg 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 118 gccttgtccc cacatattcg 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 119 aagcgaattg cattgtgtga 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 120 ggtctgcagt ttccgaatct 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 121 agagatttga ggtctgcagt 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 122 caggtaggct tcgtggattc 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 123 cccgggcctt gaccttgttc 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 124 gcgagtatga cccgggcctt 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 125 tgacaaaagg cgggttgttg 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 126 ccatgtcatg tatgacaaaa 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 127 cacaaggtct ccatgtcatg 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 128 aagaatcgga cctctgcctc 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 129 gcagcagtgg aagaatcgga 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 130 acatgcactg gcagcagtgg 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 131 gtctccacgg acatgcactg 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 132 agctccgtga cggtctccac 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 133 agcctgggat agccttggca 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 134 aagtttgcaa agcctgggat 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 135 gcttcataca caccgtactt 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 136 cgtgaagatg gcttcataca 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 137 gcgaagtcaa acttgggttc 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 138 aatgtcactg tcatccagtt 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 139 gcaaattata gcagccacaa 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 140 gatctccaca gcaaattata 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 141 gaaggccagg ccgatctcca 20 <210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 142 cctatgttta gaaggccagg 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 143 aatcccctcc tgcaacttct 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 144 agcacgtgca caatcccctc 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 145 tggttgctct gcaggtggag 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 146 gagctgcgca tgctccgtga 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 147 actcggtctt cttgatgacc 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 148 tagatctctt gcaacagtgg 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 149 catgtctctg tagatctctt 20 <210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 150 atcagtacat gtctctgtag 20 <210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 151 aggaaagatc agtacatgtc 20 <210> SEQ ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 152 tccctgctct cctgtatggg 20 <210> SEQ ID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 153 gcaaatccct gctctcctgt 20 <210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 154 tctgtgcaaa tccctgctct 20 <210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 155 cacccccatt tcggtagcag 20 <210> SEQ ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 156 ggccacacct tgacttgtag 20 <210> SEQ ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 157 gctgcgaaca ccaatgttcg 20 <210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 158 ccacgccgtg agaagggagc 20 <210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 159 tctcctctaa gttccccgag 20 <210> SEQ ID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 160 cttcaacttg gcggcagcgt 20 <210> SEQ ID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 161 tttgaaggag ctccacagca 20 <210> SEQ ID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 162 tagcgtgtgc cctctccagt 20 <210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 163 ttcaacttgg ctctcctcta 20 <210> SEQ ID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 164 ggctgcactc cgcctgcggg 20 <210> SEQ ID NO 165 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 165 ttcaacttgg ctgaggctgc 20 <210> SEQ ID NO 166 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 166 tctagatcgc acagcttgtt 20 <210> SEQ ID NO 167 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 167 cgttgagctg gtctagatcg 20 <210> SEQ ID NO 168 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 168 ttcaacttgg cggccaggac 20 <210> SEQ ID NO 169 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 169 gcgcaccggc caggactgaa 20 <210> SEQ ID NO 170 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 170 ggcgagacac accccctgga 20 <210> SEQ ID NO 171 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 171 ccctgggcac ctgaggctgc 20 <210> SEQ ID NO 172 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 172 cctctccagt ggctgtgggt 20 <210> SEQ ID NO 173 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 173 ctccagttac ctctcctcta 20 <210> SEQ ID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 174 cagcaaagcc taggctgtga 20 <210> SEQ ID NO 175 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 175 cagcacttac ctgtgatgac 20 <210> SEQ ID NO 176 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 176 aagcgaattg ctggagttgg 20 <210> SEQ ID NO 177 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 177 ccaggccgat ctacgctcaa 20 <210> SEQ ID NO 178 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 178 tagaaggcca ggccgatcta 20 <210> SEQ ID NO 179 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 179 tttgaaggag ctttgggaag 20 <210> SEQ ID NO 180 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 180 tgtggactca acagtttgtg 20 <210> SEQ ID NO 181 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 181 ctcagaactg agaagctgtc 20 <210> SEQ ID NO 182 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 182 aagagcttgg agctcggcgc 20 <210> SEQ ID NO 183 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 183 ctggtcgcga tggtggacac 20 <210> SEQ ID NO 184 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 184 tatctgaaga gttcctgcaa 20 <210> SEQ ID NO 185 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 185 gcaatccatc ggcgaggata 20 <210> SEQ ID NO 186 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 186 gaggatagtt ctggaagctt 20 <210> SEQ ID NO 187 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 187 ggaataccag tatttaggaa 20 <210> SEQ ID NO 188 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 188 tcctggctca gatggctcgg 20 <210> SEQ ID NO 189 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 189 agatggctcg gtcatcacgg 20 <210> SEQ ID NO 190 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 190 tctcccagtg gagcattgaa 20 <210> SEQ ID NO 191 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 191 catcgaatgt agaatctgcg 20 <210> SEQ ID NO 192 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 192 ctatcattac ggagtccacg 20 <210> SEQ ID NO 193 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 193 tattgtcgat ttcacaagtg 20 <210> SEQ ID NO 194 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 194 gatttcacaa gtgcctttct 20 <210> SEQ ID NO 195 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 195 tctgtcggga tgtcacacaa 20 <210> SEQ ID NO 196 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 196 tcgttttgga cgaatgccaa 20 <210> SEQ ID NO 197 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 197 gatctgagaa agcaaaactg 20 <210> SEQ ID NO 198 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 198 aactgaaagc agaaattctt 20 <210> SEQ ID NO 199 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 199 aaagcagaaa ttcttacctg 20 <210> SEQ ID NO 200 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 200 agaaattctt acctgtgaac 20 <210> SEQ ID NO 201 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 201 ctcaaatctc tggccaagag 20 <210> SEQ ID NO 202 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 202 aatctacgag gcctacttga 20 <210> SEQ ID NO 203 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 203 gaacttcaac atgaacaagg 20 <210> SEQ ID NO 204 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 204 gccaagctgg tggccaatgg 20 <210> SEQ ID NO 205 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 205 gaacaaggag gcggaggtcc 20 <210> SEQ ID NO 206 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 206 tcgcaaactt ggacctgaac 20 <210> SEQ ID NO 207 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 207 aaatacggag tttatgaggc 20 <210> SEQ ID NO 208 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 208 tcgccatgct gtcttctgtg 20 <210> SEQ ID NO 209 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 209 aagcctaagg aaaccgttct 20 <210> SEQ ID NO 210 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 210 ttgtggctgc tatcatttgc 20 <210> SEQ ID NO 211 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 211 aaaaatgcag gagggtattg 20 <210> SEQ ID NO 212 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 212 tgtgctcaga ctccacctgc 20 <210> SEQ ID NO 213 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 213 tccggcagct ggtgacggag 20 <210> SEQ ID NO 214 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 214 acatgtactg agttccttca 20 <210> SEQ ID NO 215 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 215 attttgcaca aatatccacc 20 <210> SEQ ID NO 216 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 216 ggaaacttgg gcacagaatt 20 <210> SEQ ID NO 217 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 217 aagaggtaca tacacgttta 20 <210> SEQ ID NO 218 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 218 aaatggtcac aagttctttg 20 <210> SEQ ID NO 219 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 219 ttcccgtgcc agtgccacac 20 <210> SEQ ID NO 220 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 220 ttcctcccag tagcttggag 20 <210> SEQ ID NO 221 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 221 cagtgaaaag acagtgacat 20 <210> SEQ ID NO 222 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 222 gtcaccacag tagcttggag 20 <210> SEQ ID NO 223 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 223 ctgtctgtgg tctccagcgt 20 <210> SEQ ID NO 224 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 224 gcaacatgga accagtgtcg 20 <210> SEQ ID NO 225 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 225 aagatggaaa cagttcattc 20 <210> SEQ ID NO 226 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 226 aaaaccttaa tagcttacca 20 <210> SEQ ID NO 227 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 227 aatagcttac caagtactaa 20 <210> SEQ ID NO 228 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 228 tgaagttcaa ggccctgcct 20 <210> SEQ ID NO 229 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 229 ccatccaaca tggtggacac 20 <210> SEQ ID NO 230 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 230 gcagatgacc tggaaagtcc 20 <210> SEQ ID NO 231 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 231 tggaaagtcc cttatctgaa 20 <210> SEQ ID NO 232 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 232 gagggctctg tcatcacaga 20 <210> SEQ ID NO 233 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 233 catcgagtgt cgaatatgtg 20 <210> SEQ ID NO 234 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 234 cgaatatgtg gggacaaggc 20 <210> SEQ ID NO 235 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 235 tcacacaatg caattcgctt 20 <210> SEQ ID NO 236 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 236 agattcggaa actgcagacc 20 <210> SEQ ID NO 237 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 237 actgcagacc tcaaatctct 20 <210> SEQ ID NO 238 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 238 gaatccacga agcctacctg 20 <210> SEQ ID NO 239 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 239 gaacaaggtc aaggcccggg 20 <210> SEQ ID NO 240 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 240 aaggcccggg tcatactcgc 20 <210> SEQ ID NO 241 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 241 caacaacccg ccttttgtca 20 <210> SEQ ID NO 242 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 242 ttttgtcata catgacatgg 20 <210> SEQ ID NO 243 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 243 catgacatgg agaccttgtg 20 <210> SEQ ID NO 244 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 244 gaggcagagg tccgattctt 20 <210> SEQ ID NO 245 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 245 tccgattctt ccactgctgc 20 <210> SEQ ID NO 246 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 246 ccactgctgc cagtgcatgt 20 <210> SEQ ID NO 247 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 247 cagtgcatgt ccgtggagac 20 <210> SEQ ID NO 248 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 248 gtggagaccg tcacggagct 20 <210> SEQ ID NO 249 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 249 tgccaaggct atcccaggct 20 <210> SEQ ID NO 250 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 250 atcccaggct ttgcaaactt 20 <210> SEQ ID NO 251 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 251 tgtatgaagc catcttcacg 20 <210> SEQ ID NO 252 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 252 gaacccaagt ttgacttcgc 20 <210> SEQ ID NO 253 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 253 aactggatga cagtgacatt 20 <210> SEQ ID NO 254 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 254 ttgtggctgc tataatttgc 20 <210> SEQ ID NO 255 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 255 tataatttgc tgtggagatc 20 <210> SEQ ID NO 256 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 256 tggagatcgg cctggccttc 20 <210> SEQ ID NO 257 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 257 cctggccttc taaacatagg 20 <210> SEQ ID NO 258 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 258 agaagttgca ggaggggatt 20 <210> SEQ ID NO 259 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 259 gaggggattg tgcacgtgct 20 <210> SEQ ID NO 260 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 260 ctccacctgc agagcaacca 20 <210> SEQ ID NO 261 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 261 tcacggagca tgcgcagctc 20 <210> SEQ ID NO 262 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 262 ggtcatcaag aagaccgagt 20 <210> SEQ ID NO 263 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 263 aagagatcta cagagacatg 20 <210> SEQ ID NO 264 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 264 gacatgtact gatctttcct 20 <210> SEQ ID NO 265 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 265 cccatacagg agagcaggga 20 <210> SEQ ID NO 266 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 266 acaggagagc agggatttgc 20 <210> SEQ ID NO 267 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 267 agagcaggga tttgcacaga 20 <210> SEQ ID NO 268 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 268 ctgctaccga aatgggggtg 20 <210> SEQ ID NO 269 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 269 ctacaagtca aggtgtggcc 20 <210> SEQ ID NO 270 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 270 gtcctggccg ccaagttgaa 20 <210> SEQ ID NO 271 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 271 ttcagtcctg gccggtgcgc 20 <210> SEQ ID NO 272 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 272 gcagcctcag gtgcccaggg 20 <210> SEQ ID NO 273 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 273 tcacagccta ggctttgctg 20 <210> SEQ ID NO 274 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 274 ttgagcgtag atcggcctgg 20 <210> SEQ ID NO 275 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <400> SEQUENCE: 275 tagatcggcc tggccttcta 20 <210> SEQ ID NO 276 <211> LENGTH: 1646 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 276 ggcccaggct gaagctcagg gccctgtctg ctctgtggac tcaacagttt gtggcaagac 60 aagctcagaa ctgagaagct gtcaccacag ttctggaggc tgggaagttc aagatcaaag 120 tgccagcaga ttcagtgtca tgtgaggacg tgcttcctgc ttcatagata agagcttgga 180 gctcggcgca caaccagcac catctggtcg cgatggtgga cacggaaagc ccactctgcc 240 ccctctcccc actcgaggcc ggcgatctag agagcccgtt atctgaagag ttcctgcaag 300 aaatgggaaa catccaagag atttcgcaat ccatcggcga ggatagttct ggaagctttg 360 gctttacgga ataccagtat ttaggaagct gtcctggctc agatggctcg gtcatcacgg 420 acacgctttc accagcttcg agcccctcct cggtgactta tcctgtggtc cccggcagcg 480 tggacgagtc tcccagtgga gcattgaaca tcgaatgtag aatctgcggg gacaaggcct 540 caggctatca ttacggagtc cacgcgtgtg aaggctgcaa gggcttcttt cggcgaacga 600 ttcgactcaa gctggtgtat gacaagtgcg accgcagctg caagatccag aaaaagaaca 660 gaaacaaatg ccagtattgt cgatttcaca agtgcctttc tgtcgggatg tcacacaacg 720 cttttgtcat acatgatatg gagacactgt gtatggctga gaagacgctg gtggccaagc 780 tggtggccaa tggcatccag aacaaggagg cggaggtccg catctttcac tgctgccagt 840 gcacgtcagt ggagaccgtc acggagctca cggaattcgc caaggccatc ccaggcttcg 900 caaacttgga cctgaacgat caagtgacat tgctaaaata cggagtttat gaggccatat 960 tcgccatgct gtcttctgtg atgaacaaag acgggatgct ggtagcgtat ggaaatgggt 1020 ttataactcg tgaattccta aaaagcctaa ggaaaccgtt ctgtgatatc atggaaccca 1080 agtttgattt tgccatgaag ttcaatgcac tggaactgga tgacagtgat atctcccttt 1140 ttgtggctgc tatcatttgc tgtggagatc gtcctggcct tctaaacgta ggacacattg 1200 aaaaaatgca ggagggtatt gtacatgtgc tcagactcca cctgcagagc aaccacccgg 1260 acgatatctt tctcttccca aaacttcttc aaaaaatggc agacctccgg cagctggtga 1320 cggagcatgc gcagctggtg cagatcatca agaagacgga gtcggatgct gcgctgcacc 1380 cgctactgca ggagatctac agggacatgt actgagttcc ttcagatcag ccacaccttt 1440 tccaggagtt ctgaagctga cagcactaca aaggagacgg gggagcagca cgattttgca 1500 caaatatcca ccactttaac cttagagctt ggacagtctg agctgtaggt aaccggcata 1560 ttattccata tctttgtttt aaccagtact tctaagagca tagaactcaa atgctggggg 1620 aggtggctaa tctcaggact gggaag 1646 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding PPAR-alpha, wherein said compound specifically hybridizes with said nucleic acid molecule encoding PPAR-alpha (SEQ ID NO: 4) and inhibits the expression of PPAR-alpha.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding PPAR-alpha (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR-alpha.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding PPAR-alpha (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR-alpha.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding PPAR-alpha (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR-alpha.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding PPAR-alpha (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR-alpha.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of PPAR-alpha in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of PPAR-alpha is inhibited.
 19. A method of screening for a modulator of PPAR-alpha, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding PPAR-alpha with one or more candidate modulators of PPAR-alpha, and b. identifying one or more modulators of PPAR-alpha expression which modulate the expression of PPAR-alpha.
 20. The method of claim 19 wherein the modulator of PPAR-alpha expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of PPAR-alpha in a sample using at least one of the primers comprising SEQ ID NOs: 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with PPAR-alpha comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of PPAR-alpha is inhibited.
 24. The method of claim 23 wherein the disease or disorder is a hyperproliferative disorder. 